Ecophysiological Responses of Tomato (Lycopersicon

esculentum Mill.) Under Mercury Stress.

By

Sara Aimen
Roll no: MPBOT-18-01
Session: 2018-2020

DEPARTMENT OF BOTANY
THE WOMAN UNIVERSITY MULTAN, MATTITAL
CAMPUS, MULTAN PAKISTAN
Ecophysiological Responses of Tomato (Lycopersicon
esculentum Mill.) Under Mercury Stress.

A thesis submitted to

THE WOMAN UNIVERSITY MULTAN

In partial fulfillment of the requirements for the Degree of

MASTER OF PHILOSOPHY
In

BOTANY
Sara Aimen
Roll no: MPBOT-18-01
Session: 2018-2020

DEPARTMENT OF BOTANY
THE WOMAN UNIVERSITY MULTAN, MATTITAL
CAMPUS,
MULTAN, PAKISTAN
August 2020
 CERTIFICATE
It is certified that the thesis entitled “Ecophysiological responses of Tomato (Lycopersicon
esculentum Mill.) Under mercury stress” submitted by Ms. Sara Aimen an original work of
the author and has been carried out under my supervision. I endorse its evaluation for the
award of degree of Master of Philosophy in Botany through the official procedures of The
Women University Multan, Multan.

Supervisor ______________________
Dr. Adeela Haroon
Assistant Professor

Department of Botany

The Women University Multan, Multan.

External Examiner _____________________

 DECLARATION
I ―Sara Aimen‖ hereby declares that the thesis, entitled ―Ecophysiological responses of
Tomato (Lycopersicon esculentum Mill.) Under mercury stress‖ is my own work was and
carried out by me under the supervision of Dr. Adeela Haroon, Assistant Professor, in the
Department of Botany, The Women University Multan, Multan. I also declare that the
material included in this thesis has not been used in part or full in a manuscript already
submitted or in the process of submission in partial or complete fulfillment of the award of
any other degree from any institution.

_____________
Sara Aimen
August, 2020
 DEDICATION

All my Achievements are dedicated to my Loving Family and my

Teachers

Specially Thanks to my Father Mr. Ameer omer farooqi, In the Compliance of

this Research Work

 Table of Contents
ACKNOWLEDGEMENT……………………………………………………..i

LIST OF FIGURES……………………………………………………………ii

LIST OF TABLES ………………………………………….…………….......iii

ABSTRACT…………………………………..………………………………..iv

CHAPTER I: INTRODUCTION……………………………………..…….1-6

CHAPTER II: LITERATURE REVIEW…………………………...……7-27

CHAPTER III: MATERIAL AND METHOD………………………………28-35

3.1. Experimental trial………………………………………………………..28

3.1.1 Determination of Soil Texture……………..…………………………..28

3.1.3. Preparation of pots…………………………………………………….28

3.1.2. Seed Material…………………………………………………………..28

3.1.3. Preparation of pots…………………………………………………….28

3.1.4. Soil Preparation …………………………………………...…………..29

3.2. Seed Sowing and Mercury (Hg) Treatments...……………...………..29

3.2.1 Treatment Plan……..………………………………………………… 29

3.3. Morphological and plant biomass parameters……………..………30-32

3.4. Analysis of plant Biomass …………………………………….………..32

3.9. Yield parameter of plants……………………………..……………..34-35

CHAPTER IV: RESULTS AND DISCUSSION………..………………36-64

RESULTS………………………………………………..……………..……..36

4.1. Determination of Soil Texture..................................................................36

4.2. MORPHOLOGICAL AND PLANT BIOMASS ATTRIBUTES……..37

4.2.1. Total Height of Plants………………………………………………….37

4.2.2. Shoot Length……….………………………………………………......37

4.2.3. Root Length………………….…………………………………………38

4.2.4. Numbers of Branches /Plant/Treatment……………………………...41

4.2.5. Numbers of Leaves/Plant/Treatment……………………....…………42

4.2.6. Numbers of Flowers Plant-1Treatment-1……………………………..45

4.2.7Growth Tolerance Index (G.T.I %)……………………………………47

4.2.8. Plant Leaf Area by Portable Leaf-Area-Meter...………………….....47

4.3. ANALYSIS OF PLANT BIOMASS. ……………………………….50-51

4.3.1 Determination of Fresh Weight (g plant-1)……………………………50

4.3.2 Fresh weight of shoot plant-1treatment-1………………………………50

4.3.3 Fresh weight of root plant-1treatment-1…………………………….….51

4.3.4 Fresh Weight of Total Plant……………………………………………50

4.4 Determination of dry weight (g/plant)…………………………………..51

4.4.1. Dry weight of shoot plant-1treatment-1….…………………………….51

4.4.2 Dry weight of root plant-1 treatment-1………………………………...51

4.4.3. Total plant dry weight………………..………………………………..51

4.5. BIOCHEMICAL ANALYSIS……………………………………….55-61

4.5.1. Determination of Chlorophyll Contents (SPAD Value)……...……...55

4.5.2. Uptake of Mercury Heavy Metal Determination ……………..…......55

4.6. Leaf proline contents (as stress indicator)………………………..…….59

4.7. Leaf protein (ug/g)…………………..…………………………………...59

4.8. Tomato Fruit Juice pH…….…………………………………………….59

4.9YIELD PARAMETER OF PLANTS…...……………..……………..62-64

4.9.1 Numbers of fruits per plant..…………………………………………..62

4.9.2. Weight of Fruits per Plant……………………..……………………...62

4.9.3 No. of seeds in fruit plant-1 treatment-1...................................................63

4.9.4 Fruit Measurement per Plant (width+Length)……………………….64

DISCUSSION………………..…………………………………………….67-71

CONCLUSION………………...………………………………………..........72

REFERANCES…………………………………………………………....73-90
 ACKNOWLEDGEMENT
Great ALLAH, the most gracious, merciful and beneficent has given me the strength and
make me able to fulfill the requirements of this thesis successfully in a short period of time
with in limited resources and knowledge. I like to express my deep gratitude and regards to
Dr. Adeela Haroon, Head of the Dept. for providing a healthy environment to continue the
research work.

I am lucky enough to have a supervisor, like Dr. Adeela Haroon, for her timely and valuable
help, along with healthy criticism, during all phases of development of this research. I would
like to say thank to all of my teachers, as they have been the source of guidance and
inspiration throughout the period of this degree, for me and I am grateful to all of my friends.
I would like to express my special thanks, from the core of my heart, my loving and caring
parents. Rather I feel proud, for having the GOD gifted privilege, being daughter of them.
They are really a great gift of almighty GOD, providing endless and multi dimension
transmission of love on all the modes of life, providing inspiration, motivation and
encouragement, without these prerequisite this was not possible to achieve this inspiration.

I would like to pay my special thanks to Dr. Allah Bakhsh Gulshan (Associate professor of
Botany, Ghazi university D. G khan) and my beloved uncle Malik Imran Atta (Assistant
professor of Botany) for their guidance and help during overall period of research work.

I am feeling very immense pleasure by writing these lines about my beloved brothers Abdul
Basit and Dr. Osama umer, and my friends Javeria Irshad, Tayyaba Riaz, for their moral
support.

Sara Aimen
 LIST OF FIGURES
Fig: 3.1 Clay pots filled with experimental soil placed in lawn of Ghazi University………..29

Fig: 4.1Tomato plants showing growth under various levels of Hg treatment and control….37

Fig: 4.2.Effect of various treatments of Hg on shoot length…………………………...…….38

Fig: 4.3 Effect of Hg treatments on the numbers of branches…………………………....….41

Fig: 4.4 Numbers of leaves under Hg treatment………………………………………….…42

Fig: 4.5 Numbers of fruit per treatment……………………………………………………..62

Fig: 4.6 fruit weight by digital weighing balance……………………………………………63

Fig: 4.7 seeds numbers per treatment………………………………………………………..63

Fig: 4.8 Effect of Hg on fruit size……………………………………………………………64

Fig: A effect of Hg on total height of plant, shoot length and root length of tomato plants…40

Fig: B effect of Hg on numbers on branches in tomato plants…..……………………44

Fig: C effect of Hg on numbers of leaves in tomato plants………..………………………...44

Fig: D decline in numbers of flower with increasing concentration of Hg………...………..46

Fig: E Assessment of leaf area (cm2) at all Hg levels……………………………………….49

Fig: F Presented the Tolerance index of tomato plant at Hg treatments…………………….49

Fig: G Assessment of shoot, root, and total plant fresh weight of tomato under Hg toxicity.54

Fig: H Assessment of root, shoots, and total plant Dry weight of tomato under Hg toxicity.54

Fig: I Analysis of SPAD value under phytotoxic Hg………………………………..………57

Fig: J Determination of Hg metal accumulation in root, stem and leaves……......…………58

Fig: K Demonstrates effect of Hg on leaf proline, protein, and tomato juice pH…………...60

Fig: L Presented yield parameters (numbers of fruits, weight of fruits, fruit size, numbers of
seed/fruit) affected by Hg toxicity……………………………………………………………65

ii
 LIST OF TABLES
Table: 4.1 Study of various characteristics of Soil samples…………..…………………….36

Table: 4.2 One Way Anova for Growth Parameters………………………………………...39

Table: 4.2.1 Mean values for growth parameters of tomato plants at Hg treatments….….40

Table: 4.3 One Way Anova for Numbers of Branches/Plant and Leaves/Plant…………….43

Table: 4.3.1 Mean Values for Numbers of Branches/Plant and Leaves/Plant……...…….....43

Table: 4.4 One Way Anova for Numbers of Flowers….……………………………………45

Table: 4.4.1 Mean Values for Numbers of Flowers…………………………………………46

Table: 4.5 One Way Anova for Leaf Area and Growth Tolerance Index…………………...48

Table: 4.5.1 Mean Values of Leaf Area and Growth Tolerance Index………….…………..48

Table: 4.6 One Way Anova for Various Characteristics of Plant Biomass…...……………..52

Table: 4.6.1Mean Values for Plant Biomass………………………………………………...53

Table: 4.7 One Way Anova for Plant Biochemical Analysis………………………………..56

Table: 4.7.1Mean values of chlorophyll and mercury metal accumulation…...…………….57

Table: 4.8 One Way Anova for Analysis of Proline, Protein, Leaf Color and Tomato pH…60

Table: 4.8.1 Mean Values of Leaf Proline, Protein, Leaf Color Appearance, Tomato pH.…60

Table: 4.9 One Way Anova for Yield Parameters…………………………………………...65

Table: 4.9.1 Mean values of yield parameters of tomato plants…………………………….66

iii
 Abstract

In towns of Pakistan, untreated water and the industrial effluents containing excessive
amount of heavy metals, Mercury (Hg) is one among these metals that is discharged directly
into different water system or canals. This polluted water containing Hg as major
contaminant, is used for irrigation of crops particularly vegetables and fodder. Plant crops
including vegetables accumulate Hg concentration through their root system and leaves as
well. The present research work was performed to explore the phytotoxic effect of Hg
contamination on various growth parameters of tomato plants. Tomato is most important crop
and cultivated commonly in Pakistan. Tomato seeds were obtained from Ayub agriculture
research institute, Faisalabad. Seed were exposed to different concentration (5, 10, 20, and
30mg/kg) of mercuric chloride (HgCl2) in pot soil. Various parameters including tomato
growth, plant height, length of root and shoot, biomass production, yield, and biochemical
analysis were studied under this project. A major effect of Hg contamination was observed on
the length of root and shoot. The total height of plant was reduced from 11-56% at T1-T4
level. A visible reduction was seen in shoot and root length i.e., 8-52% and 8-66%
respectively when compared with control. The branches and leaves numbers decreased to 4.7-
33% and 4.8-34% respectively. At highest dose of Hg, the numbers of flower decreased up to
63%. About 2.4-30% reduction in leaf surface/ leaf area T1-T4 at mercury level. Growth
tolerance index reduced to 11-56%.

Mercury (Hg) also had a substantial influence on biomass of plant. Fresh and dry weight of
total plant, root, shoot declined with increasing doses of Hg. Dry weight of total plant
decreased from 20-72%, while total plant fresh weight reduction was from 6 to 37%. The
proline content was increased with respect to mercury concentration; at maximum dose (30
mgHg/kg) the proline content increased upto 47%. Chlorophyll was degraded at high dose of
Hg the maximum decline in chlorophyll level was 43.8% and this lead to change leaf color.

In addition, mercury (Hg) contributed an adverse impact on yield of plant. The fruit numbers
decline in response to Hg toxicity like; 14, 28.5, 52.3 and 66.6% at T1, T2, T3, and T4 levels
respectively. Seed numbers also decreased to 13-39%. Fruit weight is significantly affected
by Hg. The decline in fruit weight ranged from 8-47%. Plants showed less effect towards
lower concentrations of Hg (5-10 mg/kg) than higher doses (20-30 mg/kg) indicating plant
resistance.

iv
 CHAPTER 1

INTRODUCTION
In recent years, heavy metal pollution in the environment has become amplified because of

diverse industrial activities, solid waste management and agricultural practices. Metal

enriched effluents also resulted in the pollution of aquatic systems. The use of such polluted

water as irrigation source pollutes the irrigating agri-land ultimately affecting crops.

Therefore, soil pollution via the food chain poses great threat to the wellbeing of humanity

(McLaughlin et al., 2009).

―Heavy metals‖ are naturally occurring metallic elements with high density superior to

5g/cm3 mainly found on earth‘s crust. Heavy metals include mercury (Hg), lead (Pb), arsenic

(As), chromium (Cr), cobalt (Co), cadmium (Cd),copper (Cu), iron (Fe), manganese (Mn),

molybdenum (Mo), nickel (Ni), zinc (Zn) and the platinum group elements (Nagajyoti et al.,

2010). Many scientists stated that heavy metals can be the source of soil and water pollution

due to human activities (Alloway, 1995; Rouphael et al., 2008). Like in the modern world

industrialization and increasing population are increasing heavy metal contamination in

biosphere (Wahid et al., 2000) and causes hazardous effects to plants, animals and humans

(Azevedo and Lea, 2005; Yang et al., 2015).

Mercury (Hg) is one of the most toxic/poisonous environmental pollutants globally. At room

temperature, it is in liquid form at room temperature and highly water soluble therefore

changes to gaseous phase enabling it to transport into different ecosystems efficiently and

deposited in soil and water bodies for prolong period. Its density is 13.69 g/ cm3 upon liquidity

and at solid state it is 14.184 g/cm3. However, 3.59% decline in volume of mercury by

freezing. Hg exists in various forms.

1
Different states of Mercury:

a) Environmental mercury (Hgo)

b) Inorganic mercury (Hg2+)
c) Mercury associated with ions (ClHg, SHg)
d) Calomel/mercurous chloride (Hg2Cl2)
e) Organic mercury (CH3-Hg)

Mercury (Hg) is being used up by humans for last 2500 years in many technological areas like

informatics, manufacturing batteries, in light bulb, paints and explosives, medicine and

cosmetics (Ahmmad, 2018) Hg is discharged into the environment and agri-land through

various sources. However, Hg is added onto the ecosystem through natural process so far,

human activities are also the main cause of Hg pollution through untreated industrial waste

materials. Owing to its toxicity, Hg is considered a universal pollutant with excessive health

concerns. (Niane et al., 2015; Dai et al., 2013; Zhou et al., 2007). It is estimated that natural

mercury emissions add two-thirds of pollution whereas anthropogenic factor releases about

one-third of pollution. Natural sources of mercury emission are erosion of metallic Hg,

weathering and sulfide ores like cinnabar (HgS). While mining, agronomic and industrial

activities are due to anthropogenic sources (Pinedo-Hernan-dez et al., 2015; Yang et al.,

2015). The environmental pollution by Hg is caused by following sources like mining and

smelting, fossil fuels burning, agriculture used in fertilizers and pesticides, manufacture

industries of paper, pulp, paint and battery, medical instrumentation, sewage sludge, silver and

gold mining (Wang and Greger 2004; Singh and Agrawal, 2007). Mercury is ranked at the 3rd

position on the earth after arsenic (As) and lead (Pb) that entered and dumped into water

canals and soil continuously then leaked into our atmosphere/environment and mixed to our

food and water (Clifton 2007). In environment the level mercury rises to three times due to

man-made activities that lead to increasing mercury burden in atmosphere to 1.5% every year

(Rice, 2014). Mercury polluted soil or the relocation of polluted water can enter into the food

2
chain via herbs/plants and livestock. Once in the food chain mercury can bio accumulate

causing adverse effects to human health (Davidson, 2004).

Mercury Toxicity to Crops

The plants can uptake metals that include both essential and non-essential by their roots and

leaves as well. Metals in vapor form enter the plant through stomata of leaves while ionic

form of metals enters through cuticle of leaf mostly (Thien-Trong, 2017). Environmental

mercury (Hgo) that exist in vapors form is entered via stomata of leaf (Azevedo, 2012). The

toxic metal ions enter the plant cells in a same way as micronutrients are taken up by them

and compete these micronutrients for absorption. Plants accumulate mercury in their roots but

some plants are capable of accumulating mercury (Hg) in shoots at adequate level either by

direct mercury absorption in gaseous form or by transportation of Hg. But Suszcysky and

Shann (1995) in a research work revealed that when plants are exposed to environmental

mercury (Hgo) it accumulates in shoots and not translocated into roots. However, heavy

metals, when they are present in soil in high concentration, inhibit the growth of plant,

metabolic, biochemical and physiological process, cause leaf chlorosis, damage the root tips,

enzymes impairment and lowers uptake of nutrients and water in wide range of plants (Sardar

et al., 2013). The mercury has impact on the seed germination, growth and yield of different

plant species due to its toxicity (Zhou et al., 2007 and Ling et al., 2010) demonstrated the

decline in the growth of coleoptile and root elongation in four vegetables Cabbage (Brassica

rapa) spinach (Spinacia oleracea), Cole (B. napus), head cabbage(B. oleracea). Mercury

hampers seed germination, causes reduction in plant growth, decreases the photosynthetic

activity, rate of transpiration water uptake and reduces chlorophyll content enzyme activity in

plants (Gardea-Torresdey et al., 2005; Yadav, 2013; Ahamad et al., 2018).

Mercury (Hg) is considered a major pollutant in soils due to its yearly import into agronomic

soil (Patra and Sharma, 2000). Hg is extremely poisonous, non-essential element and its

3
dispersal in the atmosphere is reflected to be extreme ecological problems due to its persistent

nature (Liu et al., 2010). Lacerda, (1997) established a fact that ―the universal yearly mercury

discharged into atmosphere around 460 metric tons by gold extraction in the late 1980‘s/early

1990‘s which constituted about 10 percent of the total global anthropogenic releases‖. In

addition, ―The total least discharge/year and mercury (Hg) dumping in Pakistan is ―10842‖

Kg signifies 637.76 mg per capita per year Hg exposure, which is extremely shocking

number‖ (Abbas, 2012).

The association among Hg and plants has a specific rank, because this metal is extensively

being used in seed disinfectants, composts and herbicides (Zhang et al., 2007).

In addition, mercury being a transition metal, can be a source of causing oxidative damage to

plants that leads to further formation of reactive oxygen species (ROS), resulting in the

interruption of cellular metabolic pathways and caused destruction of the cellular

macromolecules (Israr and Sahi, 2006).

Pakistan is an agronomic country, however, for irrigation purpose farmers are forced to use

sewage water due to non-availability of fresh water, and nearly around 0.033 m/ha area, for

that polluted water is used for irrigation (Ensink et al., 2004). Several scientists reported that

pollution by heavy metals like Pb, Cd, Hg, As, Ni, and Se is the most evolving problem

throughout the world (Zhang et al., 2005; Ahmad and Ashraf, 2011; Ahmad et al., 2011) and

the excessive use of metals containing industrial effluent becomes the main source of soil

toxicity (Ahmad et al., 2007; Hussain et al., 2010).

Tomato (Lycopersicon esculentum Mill.) is most important member of family ―Solanaceae”.

It is the most grown vegetable of the world with a production of ―129.650.000 tons globally‖

(Osma et al., 2012). In most part of the world is considered as essential industrial and cash

crop. (Babalola et al., 2010). Tomatoes are brought from the Andes of South America.

4
Spanish conquerors carried tomato seeds to Europe in the mid-1500s, and later transported

from Europe to South and East Asia, Africa and the middle East.

Tomato a vital cash crop of Pakistan that produces 0.566 million tons and is ranked 37th

largest producer in the world. Pakistan contributed around 0.3% yield to world tomatoes and

its insignificant share in trade internationally. In Pakistan during the period of 2014-15

approximately 150,000 area per hector was used under tomato cultivation that produced

57094 tons of tomato (FAO, 2015). Tomato is quite a short duration crop providing high

yield, so it is very attractive economically. Tomatoes offer a well-balanced and healthy diet.

Fruits of tomato are commonly consumed as salads, cooked in sauces, soup and meat, fish

dishes or consumed as paste and ketchup (Geovanelli, 2002). It contains numerous

supplements, antioxidants and secondary metabolites for example vitamins C and E, beta

carotene, flavonoids, lycopene, phenolic and organic acids which are significant for human

wellbeing (Geovanelli, 2002; Rao, 2007 and Demirbas 2010).

A natural antioxidant present in tomato is Lycopene, that beneficial in prevention of the

different types of cancer growth (Adenuga et al., 2013). Since long, continual mercury

pollution and less awareness towards mercury toxicity in plants, there is a need to understand

and realize the toxicity of Hg towards plants. Formerly, the managing and controlling

responses towards mercury bioaccumulation problems were inhibited because there is less

awareness about these metal sources, its transport mode, ways of interaction and importance

in environment. Nowadays economical and nutritional conditions, their importance

concerning to tomato plants and their increased demands in market worldwide has insisted us

to study and examine effects of mercury on tomato plants. This mainly includes possible

damages through large increase in mercury pollution which is further done by exhausts of

automobiles, excessive use of fertilizers and pesticides and irrigation done by sewage water.

5
So, by observing, the importance of this work, a research was planned to evaluate the

assessment of heavy metal toxicity i.e. mercury in tomato plant.

The main objective of the current research is to evaluate the Eco physiological responses of

mercury toxicity on various growth parameters in tomato plants.

6
 CHAPTER 2

LITERATURE REVIEW
Iqbal et al., (2014) investigated that treatment of mercuric chloride in different concentrations

(1, 3, 5 and 7 mM) on A. lebbeck plant in petri plates and studied its effect on germination of

seed and seedling growth. Lowest concentration of mercuric chloride reduced Seed

germination. when concentration of mercury increased then plant Shoot and root length

decreased considerably by 5 mM treatment. Maximum reduction was observed in seedling

34%, seed germination 51%, length of root decreased to 48% and 41% reduction in dry

weight of seedling happened at 7 mM mercury treatment. A. lebbeck showed least tolerance

percentage 51.65% produced by mercury at 7mM. High level of mercury visibly reduced

potential of seed germination and seedling vigor index (SVI).

Muhammad et al., (2015) reported the influence of various mercury treatments (1, 3, 5 and 7

mM) on growth and germination of seeds of mungbean in petri-plates. Seedlings were

germinated at 1Mm of Mercuric chloride. Seedling growth decreased at high dose. At

highest dose of mercuric chloride causes significant dry weight reduction. All treatments of

mercury decreased 42% seed germination, shoot length, root length, dry weight of seedling

70% and 66% and 47% respectively. The growth and root elongation were more sensitive to

mercury stress. The Vigna radiate seedlings indicated best tolerance at 1 mM and least at

maximum Hg dose (7mM). Negative effect was observed by mung bean seedlings towards

mercury treatment.

Ling et al., (2010) examined the mercury (Hg) impact by applying different concentrations

0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2mM on seed germination, root elongation, and coleoptile growth

of four plants including Head Cabbage (Brassica oleracea), Cabbage (Brassica rapa), Cole

(Brassica compestris) and spinach (Spinacia oleracea). All treatments of mercury increases

gradually showed reduction in coleoptile growth and seed germination. They studied the rate

7
of seed germination and growth of coleoptile reduced with increased in mercury

concentrations. The sufficient reduction was showed by all seeds of four vegetable at 0.8mM,

1.6mM and 3.2mM of mercury concentration. Brassica compestris is most tolerant species,

showed highest growth of coleoptile, germination of seeds, and root elongation. Whereas

Brassica oleracea proved to be most sensitive at high Hg concentration.

Mehmood et al., (2013) investigated the influence of heavy metals mercury and lead at doses

100 and 300mg/kg on sunflower. They use three cultivars (DK-4040, Hysun-33 and NK-278)

to study the influence of lead and mercury on seed germination, various growth

characteristics, leaf area, biomass content, carotenoids and photosynthesis. At low

concentration (100mg/kg) treatment of lead and mercury did not affect considerably on

sunflower. But as the concentration increases to 300mg/kg of both lead and mercury caused

reduction in growth and photosynthetic activity. Different cultivars showed different

responses to both metal stress. They observed that the cultivar DK-4040 is most tolerant to

metal stress than Hysun-33 and the cultivar NK-278 is sensitive to lead and mercury.

Mondal et al., (2015) investigated the impact of different doses using salt solution mercury

like (0.1ppm, 0.5ppm, 1.0ppm and 1.5ppm) on growth, nodule formation, and nitrogen

fixation of legumes and development of (Vigna radiate L) plant. When the concentration was

lowermost (0.1ppm) then it had influence on sequential growth abnormalities and seedlings.

They studied that at marginal and higher concentration level of salt solution caused

substantial reduction of seedlings and nodules. High dose (1.5ppm) of Hg causes high

tolerance, with no nodule formation.

Shekar et al., (2011) reported heavy metal mercury effect on growth and development of

tomato plant (Lycopersicon esculentum Mill). Low concentration did not showed remarkable

results on morphology; physiology and total chlorophyll were increased at lowest

concentration of mercury treatment. When the concentration of mercury increased then

8
growth and development was inhibited. High doses of mercury reduced plant height and root

length. Lowest dose 1mg/kg of Hg improved the pollen viability and it was reduced to 70%,

65% and 52% at 5, 10 and 15mg/kg of Hg treated plants respectively as compared to control

81.2%. In controls number of fruits formed was 6. The number of fruits in plants decreased to

4, 3, 2 and 1 noted at Hg doses (1.0, 5.0, 10 and 15mg/kg) treated samples respectively. Hg

also caused decline in girth and fruit weight at all treatments of Hg.

Kabria et al., (2008) examined the impact of mercury on Rice (Oryza sativa L.) developed in

pots soil of three different textures clay loam, sandy clay loam and sandy loam. Different

level of mercury treatments (0.00, 0.25, 0.50, 1.00, 1.50 and 2.00 mg kg-1) was applied on

soil. Observed Hg effect on plant height, panicle number/pot tiller numbers and yield

growing in three soils that was healthier with clay loam and in soil of sandy clay loam yield

was poorer. He studied as the concentration of mercury increases then decrease in plant

height. The formation of tiller was reduced at concentration (1.50 mg Hg kg-1) of clay soil

treatment and 1mg Hg kg-1 of sandy clay loam. The highest mercury treated soils the panicle

number was decreased by 8% in clay-loam, 20% in sandy-clay-loam, plus 18% in sandy-

loam soil. Visible decrease in grain yield started at 1.50 mg Hg Kg-1 in clay-loam and sandy-

clay-loam whereas in sandy loam soil significant reduction in grain yield was at 0.50 mg Hg

Kg-1 soil treatment. They observed when the mercury treatment was highest then the decrease

in yield was 27% in clay-loam, 22% sandy clay loam, and 27% in sandy loam soil when

compared with control. Mercury had negative outcome on grain filling process and grain

yield at high dose

Tantrey et al., (2010) highlighted phytotoxicity of heavy metals cadmium and mercury and

documented their effects on proline and total chlorophyll content in Gram (Cicer arietinum

L.) plants. The plant chlorophyll content decreased at 25mol/L and proline content increased

at both treatments (10 and 25 mol/L) of Cd and Hg. While nitrogen application decreased

9
the plants proline in response to Cadmium and mercury. They studied that chlorophyll level

decreased up to 10% and 13% at treatment (10 mol/L) of cadmium and mercury. The

decreased in chlorophyll content for cadmium and mercury at dose 25mol/L was 16% and

19% respectively.

Gauba et al., (2007) reported the toxicity of mercury on leaf biochemical attributes of tomato

(Lycopersicon esculentum Mill.) by using different concentrations of Hgcl2 0.5mM, 1.0mM,

1.5mM, and 2mM per Kg soil. They observed that when the treatments were gradually

increased to highest concentration (2.0mM) cause major decreased in chlorophyll content

(chlorophyll a reduced to 89% and 72% reduction in chlorophyll b), nitrate reductase activity

decreased about 91%, the total soluble protein content reduced to 64%, while at flowering

stage the level of nitrate 151% at 2Mm and proline content 143% increased at maximum rate.

This increased in proline content depend on age of plant along with concentration. These

parameters act as a bio indicators of environmental pollution by toxic heavy metals.

Sahu et al., (2012) investigated the influence of mercury regarding toxicity and oxidative

damage on ‗wheat‘ (Triticum aestivum L.) plants by applying different concentration 0.0, 2.5,

5.0, 10 and 25uM. Mercury reduced plant roots and shoots growth. The chlorophyll content

along with total soluble protein content was decreased at highest concentration (10 and

25uM). The highest concentration (25uM) of mercuric chloride cause 30% decreased in fresh

weight of plant. While at the lowest concentration 2.5uM the 10% protein content and 16%

chlorophyll content enhanced. But when the concentration increases to highest level of Hgcl 2

it caused sufficient reduced in the biomass and protein content. The protein reduction was

about 19% in leaves and 24% in roots.

Rolka et al., (2018) demonstrated the impact of mercury on white cabbage by growing in soil

contaminated with mercury. They used different doses of HgSO4 (0, 50, 100, 150mg/kg of

soil). The pollution caused by soil contaminated mercury affected the yield of white cabbage.

10
Mercury is not toxic at lowest concentration (50mg) it did not produced sufficient damage to

yield of aerial mass. While the increased concentrations of mercury like 100 and 150mgkg -1

caused sufficient reduction in aerial mass yield (14% reduction at 150mgkg-1). They studied

that when the concentrations like 50mg and 150mg/kg of soil were applied, the root mass of

cabbage decreases. They found that when the mercury contamination in soil increases then

this caused the increase in translocation of mercury to aerial mass of cabbage. Thus increase

in concentration in aerial mass of cabbage lead to destruction of yield of cabbage.

Liu et al., (2010) evaluated the impact of mercury on wheat plant using cultivar Jinan No.17.

They investigated the toxicity of HgCl2 at wheat raised in soil contaminated with various

mercury treatments (0, 100, 200, and 500mg Hg/kg). The plant growth was influenced both at

high and low concentrations during initial or early germination stages. They studied the

chlorophyll content was promoted at low and high concentration but was inhibited at later

stages of germination. They investigated that the high level of mercury increased the

concentration of calcium along with mercury. Furthermore the phytotoxicity of mercury

influenced on calcium of leaves in winter wheat.

Ahmad et al., (2018) highlighted mercury influence on uptake and translocation and its

remedial potential of four plant species grown on mercury contaminated soil. They

investigated the phytoremediation effect of Tridax procumbens, Dodonaea viscosa, Ruellia

tuberosa and Azadiracta indica. They examined the content of mercury in plant root and

shoot. Reduction in shoot length of Tridax procumbens and Dodonaea viscosa as the days of

contamination increased (at 60th day). But in Ruellia tuberosa and Azadiracta indica show

any substantial decline in shoot length was not observed. The mercury causes declined in

fresh and dry weight of plant. They studied the bio concentration factor and translocation

factor. By their both values the plant species Tridax procumbens showed that when the bio

concentration factor enhanced the roots have greater potential to uptake and translocate

11
mercury than shoots. According to values of translocation factor Ruellia tuberosa, Dodonaea

viscosa and Azadiracta indica showed that mercury is translocated from roots to shoots

(aerial parts of plants). They found that mercury pollution has impact on plants parameters

like decreased in vegetative growth, photosynthetic activity and increased in proline and

malondialdehyde content as the days of contamination increased.

Alonso et al., (2019) evaluated phytotoxicity of Hg on plant Quercus ilex L. (holm oak).

They use nutrients solution for watering the seeds and seedling containing different mercuric

chloride concentration 0, 5, 25, and 50uM. The phytotoxicity of mercury is totally depend on

its bioavailability in soil where the plant grow. They examined the impact of mercury on

growth and germination, accumulation of mercury and nutrients in roots stems and leaves

during different stages of plant growth. They found that the morphology of roots was

changed completely but it did not have damaging effects on growth, germination and

nutrients accumulation. The variation in mercury accumulation depends on concentration of

mercury, plant organ, time and growth stage of plant. They studied that highest concentration

of mercury was present in roots after leaves and stems by comparison of different plant

organs.

Saraswat et al., (2018) reported the toxicity of mercury (heavy metal) to wheat crop (Triticum

aestivum L.) Using four varieties. In order to find tolerance behavior of these 4 varieties,

they studied GP (percentage of germination) and SGI (Speed of germination index). They

found when the mercury (Hg) concentration increases it has negative impact on germination

of all four varieties (V1 Lok-1, V2 UP-2338, V3 PBW-154 and V4 PBW-502). High mercury

concentration reduces the germination. The germination was inhibited at high concentrations

(100, 200, and 300ppm) in all wheat varieties. They recorded the ―lowest percentage of

germination in V1 variety was 23.66% and highest GP 26.66% in V4 during 24hours. While

lowest GP in V1 was 51% and highest GP 54.66% in V4 during 48hours. At 72 hours GP was

12
61.33%(lowest) in V1 and 66.66% (highest) in V, but during 96hours 82% (lowest) in V2 and

84.33% (highest) in V3 & V4. Whereas 120 hours 86% (lowest) in V2 and 94% (highest) in

V3‖. During 24 hours in all wheat varieties high concentration (100, 200, and 300ppm) of Hg

inhibit the germination. The obtained data when compared with control revealed the

maximum percentage of germination (GP) at 100ppm in V2 variety was 48.98% and

minimum GP at highest concentration (300ppm) in variety V2 was 18%. While the data

recorded from speed of germination index at concentration of mercury 100ppm in V1 43.08%

(highest) and in V2 GPI was 13.03% (lowest) at treatment of 300ppm mercury.

Sadej et al., (2019) experimented the influence of mercury contaminated soil in oat plant

(Avena sativa L.) treated with different concentrations (0, 50, 100, and 150mg Hg/Kg of soil).

In order to lessen mercury pollution they added zeolite, lime, and bentonite. High treatments

of mercury inhibit the growth and reduced roots. It also cause reduction in the aerial mass

yield. the soil contaminated with mercury reduces the yield of grain about (10.03 to 12.02%)

in Avena sativa L. the yield of grain in soil polluted with mercury was improved (1.9-11.3%)

in the presence of bentonite. the impact of both lime and benotite as compared to zeolite are

helpful to lessened the mercury phytotoxicity and positive effect on plant yield. the plants

grown in Hg contaminated soil (high concentration) showed enhanced Nitrogen (N) and

potassium (K+) content in them caused reduction in the phosphorus (P) level.

Ancient gold miners are main sources of mercury pollution in soil. Mercury is dangerous for

human and environment. According to Negrete et al.,(2016) The metal accumulated plant,

Jatropha curcas used for phytoremediation of toxic Hg is favorable to these gold mining

sites. They experimented the jatropha curcas in mercury containing hydroponic cultures.

They used the treatments 5, 10, 20, 40, 80μg Hg/mL. Mercury has impact on photosynthesis,

inhibit growth and developmental process, reduced leaf area, and biomass loss. They

examined and evaluated the total uptake of mercury, accumulation, and translocation factors.

13
The mercury uptake in hydroponic cultures in root and tissues of leaf is depending on its

concentration. In roots the accumulation of mercury was higher than leaves (7 folds) and

shoots(12 folds). At highest treatment 80μg Hg/mL net photosynthesis was inhibited to >60%

and growth inhibition was >50%.the growth of plant and rate of photosynthesis were down to

88%-95% at high concentrations of mercury 40 and 80 μg.

Meng et al., (2010) studied and evaluated the ―inorganic mercury and methyl mercury

distribution‖ in whole rice plants (Oryza sativa L.) grown at different places in soil

contaminated with mercury. The rice grown in mining area contained the high concentration

of mercury (IHg and MeHg) than control sites. Mercury polluted soil is due to activities like

excessive mining of mercury and smelting. Inorganic Mercury is found in aerial parts of

plant. Its main source of is air. However roots containing inorganic mercury (IHg)

concentrations depend on paddy soil concentrations of mercury. High concentration of

Methyl mercury is accumulated by seeds of rice than other tissues. Paddy soil is the main

source providing methyl mercury to rice plant tissues. They reported that the methylation of

―newly deposited mercury is relatively more easy than old mercury in soil‖

Liu et al., (2017) conducted pot experiment for four plants Aloe vera, Setcreasea purpurea,

Chlorophytum comosum, and Oxalis corniculata of herb and highlighted the impact of

mercury and growth agent on their growth. They studied effects of different solution of Hg

treatments & growth agent on plants ―height‖ and yield of ―biomass‖ and RGR (relative

growth rate). They examined the maximum growth of two herbs (Aloe vera and

Chlorophytum comosum) when treated with water (control). But the herb (Setcreasea

purpurea) treated with Fertilizer (Lvyebao) showed extreme growth rate. The herb Oxalis

corniculata is most resistant species to Hg treatment and they showed greatest growth rate of

aerial part of herb (Oxalis corniculata) by applying ―water and green cake fertilizer‖. While

the Chlorophytum comosum is most sensitive to Hg treatment than other species.

14
Mercury is a pollutant which is extremely toxic for environment its concentration is

increasing day by day because it is not degradable so it is very difficult task to remove

mercury because of persistent nature. Pérez-Sanz et al,.(2010) examined a plant Silene

vulgeris is known for the stabilizing effect of Hg known as ―phytostabilizer‖ that can

accumulate mercury in their organs especially in roots also called ―facultative metallophyte‖.

The efficiency of Silene vulgaris was assessed by conducting pot experiment using two soils

(mercury polluted soil and non-polluted soil) for growth of Silene vulgeris under rain shelter

throughout duration. They used two mercury treatments Hgcl2 0.6 and 5.5 mg/kg of Hg in

metal containing soil and non-contaminated soil. At low concentration of mercury the growth

of plant was healthy with no apparently decreased in biomass. But at high concentration of

mercury in soil the uptake of mercury by plants will be increased. The Silene vulgeris

accumulated high concentration of Hg in their root as compared to shoots. So they are

regarded as helpful plants in improving metal contaminated soil.

The excessive release of Hg (mercury) and Se (selenium) in environment are due to excessive

anthropogenic (industrial and agriculture) activities. Bian et al., (2016) reported the

contamination of soil by both mercury (Hg) and selenium (Se) is of concern for agricultural

yield of crop. They experimented the effect of mercury and Selenium (Hg-Se interaction) on

Brassica rapa roots in hydroponic solution by applying both Hg and Se treatments like

(mercuric chloride 1μM and sodium selenite 4μM) in combined form. These

Mutual/combined treatments of Hg-Se has impact on physiological (root growth was

inhibited, reduced biomass of roots) and biochemical parameters (the increased production of

ROS and malondialdehyde accumulated that further reduced integrity of membrane).

Furthermore the glutathione with high concentration was found in root tips. Thus they

evaluated the treatments Hg+Se in combined form were toxic for roots of Brassica rapa

causing oxidative injury and cell death in B. rapa species.

15
 Pirzadah et al., (2018) examined the mercury phytotoxicity at various concentrations 0, 25,

50, and 75mM for 15 and 30 days in Tartary buckwheat. The mercury showed its effects on

growth parameters. The level of Biomass decreased at 75uM seedlings 37.5% during 15 days

while the decline in biomass of seedlings was 67.97% at 30 days of mercury treatment. At

highest dose (75uM) during 15days of treatment the level of lipid peroxides increased

significantly 65.58% and in next 15days it was only 23.53%. The mercury also had effect on

photosynthetic activity it reduced the chlorophyll content at 15 days (42%) and (30.7%)

reduction as comparison with control at 30 days of highest mercury dose (75uM) but the level

of carotenoid and anthocyanin were elevated. At 75uM the leaf proline level depend on

concentration of mercury at 15 days the enhancement was 158.4% but during next 15days the

results was opposite the total proline level reduced to 51.35%. high concentration of mercury

was found in roots than shoot and reduced the length of both root and shoot. The antioxidant

enzymes activity like GR (glutathione reductase), APX (ascorbate peroxidase), SOD

(superoxide dismutase), POD (guaiacol peroxidase), GST (Glutathione-s-transferase) and

superoxide dismutase (SOD) showed positive correlation with mercury treatments excluding

SOD, reduced at 75uM of mercury concentration during 30 days old seedlings. There existed

positive correlation of activity of CAT (Catalase) 25uM (30.62%) to 50uM (85.47%) of

mercury treatment at 15days while activity was decreased at 75mM mercury stress for both

15 and 30 days.

Su et al., (2005) investigated the effect of atrazine from nutrient solution and mercury (Hg) on

rice plant seedlings. The nutrient solution with or without mercury was applied for 96hours

duration. They examined that both atrazine and mercury (Hg+2) reduced/inhibited growth of

seedlings and have toxic effects on seedlings of rice. The rice seedlings when applied with

1.0mg/L of mercury (Hg+2) containing nutrient solution the biomass seedlings were reduced to

50%. Up to 80% reduction in biomass of seedlings by applying 12.0mg/L atrazine. While the

16
seedlings were exposed to nutrient solution containing both atrazine (ATR) and mercury (Hg+2)

then rice seedlings promoted. The mercury concentration 0.1mg/L and 1.0mg/L in solution have

declined the uptake of atrazine by seedlings, that effect was not depending on atrazine

concentrations 6.0 and 12.0mg/L. Atrazine create uneven ―small to moderate‖ variations in

mercury (Hg+2) treatment containing rice seedlings. Their findings showed that the atrazine

(ATR) and mercury (Hg) uptake by rice seedlings are not dependent on each other.

Niu et al., (2011) experimented impact of ―Field open top chambers (OTCs)‖. And also

experimented on how wheat accumulates mercury in soil and air. They also used maize for this

purpose. They also estimated the amount of mercury level in the leaves, and found that total

level of correlated p<0.05 mercury concentration present in air. It is not sufficiently correlated to

mercury concentrations present in soil. This signified that crop foliage contained mercury from

air chiefly. The roots containing mercury (Hg) concentrations were associated with mercury

(Hg) concentrations (p<0.05) of soil generally while it is in significantly interrelated with

mercury concentrations of air. It specified that mercury in roots of crops was from the soil

essentially. There exist no significant correlations between concentrations of mercury Hg in

stems and those in air and soil. Yet, the concentration of mercury (Hg) in upper part of stems

were higher generally to those in lower /bottom portion of stems, that indicated the mercury in

air have impact stronger on stem than mercury present in soil on accumulation of mercury in

stem.

Du et al., (2005) investigated mercury (Hg) metal impact when exposed to rice Oryza sativa L.

The rice seedlings were grown in solution. They studied the relation between uptake of mercury

and arsenate. The nutrient solution containing high concentration of mercury caused reduction in

biomass of root and shoot. The biomass of roots decreased to 50% at high concentrations

1.0mg/L and 2.5mg/L. while plant biomass of shoot reduced to 50% when the concentration

0.5mg/L applied. while arsenate at 0.5 mg/L had no reduction in the yield of rice. Rice roots

17
contained mercury (Hg) concentration, therefore at 2.5mg/L of mercury the concentration factor

was 1900. Slightly enhanced in concentration of mercury was mainly by the addition of

arsenate. The high level of Hg in the solution caused enhanced the arsenate root and shoots

level. But when there is more increased in mercury level at 2.5mg/L the arsenate level

improved/increased.

Mellem et al., (2012) determined the effect of mercury (Hg), nickel (Ni) chromium (cr), lead

(Pb), Arsenic (As) and copper (cu) on the plant of Amaranthus dubius a nutritious plant grown

widely in Asia, Africa, and South America. It is leafy crop produced high yield due to its rapid

growth. This vegetable has potential to remediate heavy metals because of its hyper

accumulating activity. Amaranthus dubius developed in controlled environment in tunnel house

transferred the heavy metals like Cr, Hg, As, Pb, Cu, and Ni to other organs from roots. Bio

concentration factor (BCF) and translocation factor (TF) were calculated to examine

phytoremediation of plants. The treatments used were 25ppm, 75ppm and 100ppm of heavy

metals (Cr, Hg, As, Pb, Cu, and Ni) for four days. Low concentration 25ppm A. dubius is

capable of accumulating and translocation of Mercury (Hg), Chromium (Cr), and Lead (Pb) to

shoots from roots but when plant exposed to (75ppm and 100 ppm) higher concentrations metals

are chiefly stored in roots. Thus they reported that only Arsenic accumulate at all

concentrations representing A. dubius showed less capability of bioaccumulation at high

concentration of chromium, mercury, nickel, lead and copper In the case of As accumulation at

all concentrations.

The mercury (Hg) and arsenic (As) metals are considered the most poisonous metals. When they

becomes a part of food chain and hazardous for humans, animals and environment because they

accumulate continuously over time and increased toxicity. There are some plant species known

for removing contaminants (pollutants) form soil and water. So Comino et al., (2009)

investigated the ability of plant species Poa annua for metal accumulation and detoxification of

18
pollutants (heavy metals) and being a source of fodder for farm and wild animals and its

remediation potential related issues were studied because Poa annua involved widely in food

chain. They exposed the plant with both metals using three different treatments. Treatments of

Arsenic (As) are (0.25, 0.5, and 5mL-1) while for mercury treatments are (0.1, 0.2 and 2mgL-1).

They observed during testing phase that there was no significant change/difference in absorption

of different concentration of mercury (Hg) and Arsenic (As). The absorption of mercury and

arsenic in plant species increased with increasing concentrations.

Bhanumathi et al., (2005) reported the influence of mercury on the growth of seedling of

peanuts Arachus hypogea L. The mercuric acetate at the concentrations of 0.0001Mm and

0.001mM have impact on root and shoot length. Also on total chlorophyll contents like chl a and

chl b Mercury acetate treatments(0.0001mM and 0.001mM) have negative effect on total

protein, sugar and lipid content as well. The reduced in root length was 14.4cm (control) to

9.83cm at treatment 0.0001mM while at 0.001mM root length reduced to 7.51cm from 14.4cm.

The more decreased in root length than shoot length 10.88cm (control) to 5.84cm at 0.001mM.

But high concentration (0.001mM) of mercury reduced the shoot length to 5.67cm. Significant

chlorophyll contents decreased at both concentrations.

In West Lombok-Indonesia, due to frequent discharge of gold amalgamation tailings in

agricultural lands caused reduction in yield of maize crop in that area. Phytoremediation can

speak to a minimal effort option in contrast to ordinary methods, for example, soil expulsion.

Muddarisna et al., 2013 investigated the capacity of phytoremediation of Lindernia crustacea,

Paspalum conjugatum L., and Cyperus kyllingia Endl. in Hg contaminated soil in relation to the

ammonium thiosulphate to phytoextract/remove mercury and its influence on maize

development. They cultivated the seedling in pots for 9 weeks. The pots were filled with

mercury polluted soil, which had 15 kg capacity. For 3 plants, these treatments were examined.

Ammonium thiosulphate was applied one week before plant collection. After 9 weeks, plant root

19
and shoot had harvested. These samples then examined after that. Remaining of soil which was

contaminated with mercury used to grow maize plants. Outcomes revealed that the addition of

ammonium thiosulphate have impact to increase the concentration of mercury in plant shoots

and roots. 62% remediation was carried out without ammonium thiosulphate.

Jamal et al., (2006) reported Hg toxification on the two varieties of wheat Triticum aestivum

(Blue Silver and T. aestivum var. Punjab 85). They exposed the seeds with different

concentrations (25, 50, 75 and 100ppm) of mercuric chloride (Hgcl2) solution and results were

carried out on germination of seed and its growth. The 2 varieties upgraded the germination of

seeds in all the Hg levels. In any case of shoot, root, and seedling length, the negative results

were found. In Punjab 85 variety at highest concentration of Hgcl2 there was highest decreased

in shoot length 5.0cm that was sufficiently different in contrast to control 12.06cm. While at

100ppm of Hgcl2 both varieties of wheat showed maximum decreased in length of roots 3.90cm

blue silver variety and 3.23cm in Punjab 85variety. At high concentration (75 and 100ppm) the

seedling length of wheat varieties Blue Silver, (9.93 cm) and var. Punjab (85, 8.23 cm) reduced

significantly. Root length showed maximum reduction than shoot length and seedling length.

Blue silver had proved tolerant one.

The heavy metal Mercury (Hg) is considered as one of the major toxic pollutant because it has

capability to bio accumulate and biomagnifies in human and animal bodies through food chain.

Plants are the source of removing toxic heavy metals from soil and environment therefore they

are now used as removal agents for toxic pollutants. Malar et al., (2015) evaluated the impact of

toxicity of mercury (Hg) for plants Sesbania grandiflora. The seedlings of Sesbania grandiflora

were exposed for 10 days to different treatments of mercuric chloride (Hgcl2) 10, 20, 30, 40, 50,

and 60mgL-1. The seedlings of S. grandiflora exposed to different treatments of mercuric

chloride (HgCl2) after 10 days exposure resulted the inhibition of both root and shoot

development. The greater effects were showed by Shoots as compared to roots. The seedling

20
growth/development was reduced to 47% in shoots and 56 % for roots at highest concentration

(60mgL-1). At 40mgL-1 mercury treated plants did not show any observable toxic symptoms.

Seedlings developed at highest concentration (60mgL-1) showed 19% reduction in Biomass in

contrast to controls. The Relative water content (RWC), is considered as an indicator of heavy

metal toxicity which is reduced to 5% at highest concentration 60mgL-1. The increased total

chlorophyll content of Seedlings treated with 60 mg L-1, chlorophyll-a 92 %, chlorophyll-b

increased 76 % and increased in carotenoid contents as compared to controls was 69 %.

Sasmaz et al., (2016) studied on mercury (Hg) take-up and transport from the soil to various

organs of plants. And reported the accumulation and distribution of mercury (Hg) in 12

terrestrial plant species (roots + shoots), growing normally in soils surface of the Gumuskoy Pb-

Ag mining area. For investigation of mercury content the plant samples and their related soils

were gathered and examined by ICP-MS. The outcomes showed that examined plants samples

kept mercury (Hg) in their roots and prevented mercury transfer to aerial plant parts. The mean

enrichment factors was <1. It was 0.06 of roots (ECR) and for shoots (ECS) was 0.09. The

capacity of plant to move Hg from the roots to the shoots was showed by mean TLF values.

Moreover, with higher ECR and ECS the movement was increasing proficient.

Gupta (1998) experimented the potential of submerged aquatic macrophyte Vallisneria spiralis

plant to accumulate mercury and effect of toxicity on it in laboratory. Various concentration of

mercury 0.5, 1.0, 5.0, 10, and 20mM was exposed to young plants for 24, 48, 96, and 168hours.

At highest concentration of mercury (20mM) in plants leaf accumulated 0.25mmole per gram

dry weight and 1.12mmol per gram dry weight in roots after 168 h. As the mercury (Hg)

concentrations and treatment durations increased, it caused sufficient reduction in content of

protein, chlorophyll, and phosphorus, nitrogen, potassium. Also in vivo nitrate reductase

activity reduced at all concentrations of mercury. Leaf and root cysteine content raised at low

level of mercury and decreased at high doses. The outcomes revealed that plant had great

21
potential to uptake mercury via roots and acted as an indicator of environmental metal

contamination, in spite of the fact the mercury have negative impacts on plant physiology, and

transferred to different trophic level through food chain.

Xun et al ., 2017 evaluated the impact of mercury (Hg) on plant Cyrtomium macrophyllum by

growing the plants in mercury polluted soil of mining area at concentration of 225.73mg Hg/kg

of soil and in soil contaminated with different concentrations of mercury (HgCl 2) 5, 10, 20, 50,

100, 200, 500, and 1000mg/kg in pots. Cyrtomium macrophyllum grown at 225.73mg/kg

naturally the 2.62 was the translocation factor. Plant aerial parts contained high level of mercury

(36.44mg/kg). Pot experiments showed that Cyrtomium macrophyllum was hyper accumulator

plant could develop in high Hg concentration 500 mg/kg of soil inhibited growth without any

significant toxicity. Plant at high concentration (200mg/kg) indicated it had excellent bio

concentration factors and translocation >1. At lower concentration of Hg, presented a good

metal Hg accumulating plant. Moreover, the leaf tissue of plant presented great resistance to

mercury toxicity that enhanced the activity of super oxide dismutase and glutathione and proline

accumulation that favor movement of Hg to shoots from roots to tolerate high concentration of

soil mercury (Hg). So plant species can be beneficial remediation of mercury metal polluted

soils.

Umadevi et al., 2009 investigated the effect of mercury in plant Vigna unguiculata (cowpea).

This plant was given various treatments in order to see the response of Hg on plants (Hg) 0.05,

0.10, 0.50, 1.0, 1.5, and 2.0 mM in petri plates. The phytotoxicity of mercury to germination,

early growth of seedlings, metabolite level, heavy metal content, enzyme activities in cow pea

seeds were investigated. Mercuric chloride not only reduced germination percentage,

seedlings/radical growth, dry weight of roots and shoots at lowest concentrations (0.05-1.5mM),

also caused inhibition of growth at 2mM of HgCl2 At all low concentrations (0.05-2mM)

reduced the percentage of germination from 96% (control) to 2% at 2mM. at lowest

22
concentration of HgCl2 decreased the roots and shoot dry weight 1.2 and 1.9 respectively. as

compared to control contained 6 and 8.1 root and shoot dry weight, at 2mM decreased the roots

and shoot dry weight had 0.01 and 0.02. Roots failed to germinate at 2mM. The radicle failed to

germinate at 2mM concentration. So the seed germination was more resistant to mercury (Hg)

stress as compared to seedling growth. The observable changes in enzyme activities and

biochemical responses of Vigna unguiculata showed that they are sensitive to metals toxicity

and used as an indicator of metals stress.

Cargnelutti et al., (2006) evaluated the impact of exogenous mercury (HgCl2) on cucumber

(Cucumis sativus L.) seedlings. Mercury influenced on antioxidant enzyme, lipid peroxidation,

protein oxidation and chlorophyll content was time dependent. Different concentrations of

mercuric chloride HgCl2 (0, 0.5, 50, 250, 500uM) were applied on Cucumber seedlings for 10

and 15 days. Roots absorbed mercury and accumulated it in higher amount than shoot. The

decreased in root and shoot length was time and concentration dependent. Low concentration

(50uM) of HgCl2 in 15days old seedlings root fresh weight increased, and decreased for high

concentrations (at 250uM 96% and 98% at 500uM root length) at 10 and 15days seedlings. The

fresh biomass of root and shoot of 10 days old seedlings was decreased. Shoot fresh biomass did

not show significant reduction at 50uM in 15 days seedlings the increased in roots Dry weight at

500uM at (10 and 15 days) old seedlings, while for 250uM of HgCl2 dry weight increased only

at 15 days. All concentration of mercury produced effect on shoot dry weight. Seedlings

exposed to mercury showed high levels of lipid peroxides, increased protein oxidation levels,

and decreased chlorophyll content (59% for 10 days old seedlings and 94% for 15 days old

seedlings at 500uM), when exposed to 250 and 500uM of HgCl2. The 500uM significant

inhibition of seedlings and oxidative stress caused damage in seedlings.

Yemini (2001) examined the impact of heavy metals over Vigna ambacensis L. plant the effect

of various concentrations 0.05, 0.10, 0.50,1.00, 2.00, 5.00, 10.0, and 50.0 mM of all metals (Hg,

23
Cd, and Pb) were investigated. High concentration of heavy metals decreased growth of

seedlings, and seed germination, shoot length and radical length treated with mercury, lead, and

cadmium.) The inhibition of growth parameter depend on heavy metals concentration. Seeds

exposed to mercury (Hg) at 0.5Mm showed 16% while same concentration (0.5mM) of

cadmium (Cd) 69%, and Lead (Pb) 66% germination percentage. Germination of seed was

stopped at high concentration (10mM) of both mercury and cadmium while for lead (Pb) it

stopped at 50.0mM revealed that mercury is most toxic than cadmium and lead metals. Roots

germination was inhibited than shoot at Cadmium and mercury concentrations 1.0mM and 5mM

respectively. While Seedling growth was more sensitive to Cadmium than mercury Hg and Pb at

lowest concentration 0.5mM. High concentration of metals was examined in roots than shoot.

Palanisamy et al., 2012 investigated the outcomes of mercuric chloride (HgCl2) on morphology

and cytotoxicity of soybean (Glycine max L.) at different concentrations 0.0, 1.0, 2.0, 3.0, 4.0

and 5.0 ppm. The high concentrations of mercury (HgCl2) decreased the morphological/ growth

parameters of seedlings like germination percentage, length of root and shoot, fresh and dry

weight. The germination was totally stopped at concentration of 5.0 ppm of mercuric chloride.

Also mercuric chloride influenced cytomorphological changes in cells of root tip of Glycine max

L. like increased in chromosome aberration percentage and mitotic indices with increased in

mercuric chloride (HgCl2) concentration. At high mercury concentration the chromosomal

length (Absolute and Average) reduced progressively and diploid (2n) number of chromosome

was changed significantly. So the high concentrations of HgCl2 had critical cytotoxic and

mutagenic effects on soybean root tip cells.

Rodríguez et al., 2009 evaluated the effect of mercury [as Hg(CH3COO)2] and Au (KAuCl4)

on plants of Chilopsis linearis were grown hydroponically with mercury treatments 0, 50, 100,

and 200M Hg and Au treatments 0 and 50MAu. Seedlings were exposed to each treatment or in

combination form (Hg + Au). The outcomes revealed that when the seedlings were exposed to

24
50M Au+50M Hg root growth was inhibited to 25% and while reduction in root growth at

50MAu+100M Hg was 55%. The element uptake determination by means of ICP/OES revealed

that mercury at 50 and 100M (with and without Au) increased the sulpher concentration in

leaves. In contrast, iron (Fe) concentration increased in the plants roots treated with Au-Hg.

Moreover, plants stems exposed to mercury treatment at 100M (with Au had 239 mgHgkg-1 and

without Au had 876mg Hgkg-1). Likewise, mercury (Hg) at 50M Hg, accumulated in stem with

Au 375 mgHgkg-1and without Au 475 mgHgkg-1 d wt. At treatment (50M Au+100M Hg) higher

concentration of mercury in leaves was 287 mgHg/kg d wt. Leaves Hg accumulation was

reduced to 75 mgHgkg-1 of dry weight Without Au. Hazardous effects caused by Hg in cortex

cells and the vascular system were lower in plants treated with 50M Au+50M Hg contrasted

with plants exposed to 50M Hg. Additionally, the SEM micrographs showed Au–Hg particles

deposited inside the root. Although the mercury concentrations used in experiment showed

different degree of phytotoxicity, the plants showed great agronomic worth.

Ansari et al., 2009 experimented on plants Indian mustard (Brassica juncea L. Czern and Coss.)

cv. Pusa Jai Kisan hydroponically to investigate the impact of different concentrations 0, 5, 10,

25 and 50mM of mercury (Hg). Mercury exerted negative effects on leaf area, root and shoot

length, and on dry weight. The mercury impact depends on concentration. High level of mercury

enhanced mercury content in shoot as well as oxidative stress (MDA and H2O2). The high

concentration of mercury (50mM) caused maximum decreased in plant growth along with

increased in antioxidant enzymes activities. The growth changes influenced by mercury lead to

increase in lipid peroxidation.

Mercury contamination in grounds has become a possible danger to man wellbeing. Wheat

cultivars grown in grounds with low mercury concentration in minor or average mercury

contaminated fields are a productive ensuring safety of food. Liu et al., (2017) examined the

mercury resistance and accumulation characteristics by performing pot experiment at

25
concentrations of 1 and 5mgkg-1 for thirty general cultivars in chief wheat growing areas of

China to evaluate the mercury. Outcomes revealed at low and high concentrations of mercury (1

and 5mgkg-1) height of plants increased. Low concentration of mercury (1mgkg-1) increased

peak length of internode but reduced no. of spikelet. High mercury level (5mgkg-1) reduced the

yield parameters such as total number of grain i.e. fresh grain yield and dry grain yield. The

mercury accumulations in grains showed a significant negative correlation of plant height, spike

length and fresh grain yield in response to mercury stress. Criteria for selecting wheat resistant

to mercury were built up, which incorporated the coefficients of mercury resistance (effective

tiller number, fresh grain yield, and dry biomass) at lower Hg level and the mercury resistance

coefficients of dry grain yield and biomass towards great mercury stress. Finally, Liangxing-99,

Nongda-3163, and Gaocheng-8901 were isolated for extraordinary protection from Hg and low

accumulation of mercury. These wheat cultivars could be grown in grounds contaminated with

lesser and moderate mercury to attain nontoxic grains.

Mushtaq et al., (2018) applied the ―aqueous extract of Pongamia pinnata (PALE)‖ on zea mays

seedlings contaminated with mercury (HgCl2) in order to lessen the phytotoxic effect of

mercuric chloride (HgCl2). Mercury (HgCl2) effected the growth and germination of seedlings.

Soil contaminated with mercury concentrations (1 and 0.5mgkg-1) were used to produced stress

in seeds. Before sowing maize seeds in pots containing mercury contaminated soil they were

exposed to 5 and 2.5% PALE. The seeds (without soaking in PALE) at 1mgkg-1 of mercury

treatment showed sufficient decreased in percentage of germination, photosynthetic pigment,

root dry weight. But maize seeds that were soaked in PALE resulted in reduced phytotoxic

effects of mercuric chloride on growth and germination percentage. Therefore it act as a shield

that defend maize plants from unfavorable impacts of mercury and can be suggested for

application in Hg polluted zones of agri-lands.

26
Israr et al., 2006 investigated the impact of mercury (Hg) accumulation in seedlings of Sesbania

drummondii. The seedlings were exposed to 10, 20, 30, 40, 50, and 100mgL-1 concentrations of

mercuric chloride (HgCl2).The growth of plant, antioxidative activity along with photosynthesis

was influenced by mercury (HgCl2). The Mercury accumulation in plant tissues (shoot + roots)

increased with increasing mercury concentration. At highest level (100mgL-1Hg) roots contained

mercury in high concentration (41403mg Hg/ kg of dry weight) as compared to shoots (998mg

Hg/ kg of dry weight). The growth of plant was reduced to 36.8% at 100mgL-1 concentration of

HgCl2. The concentration of 100mgL-1 also influenced the photosynthetic activity.

27
 CHAPTER 3

MATERIALS AND METHODS

For the current research work, a trial was conducted in the wire-net-house of Department of

Botany, Ghazi University, Dera Ghazi Khan during October, 2019 - March, 2020.

3.1. Experimental Trial

3.1.1. Determination of Soil Texture

Before starting the experiment, the texture of soil was analyzed by ―X-Ray

spectrophotometer (PW 1660-Philips)‖ at Cement Company D. G. Khan to investigate the

soil to be used for pot experiment. A 10 gram of soil was strained, squashed pelletized. After

that the pellet was moved to X ray machine. In order to classify, the experimental soil

obtained values were matched with International chart of soil texture.

3.1.2. Seed Material

The experimental plant, Tomato a member of family Solanaceae, and is preferred as essential

vegetables of the world and Pakistan. Certified seeds of tomato cultivar Roma were taken

from the Ayub Agriculture Research Institute Faisalabad. Roma is most cultivated variety in

the Punjab. To start experiment disease resistant seeds with uniform size and weight were

selected and sterilized with 0.1% HgCl2 then transferred these seeds into distilled water to

eliminate the mercuric chloride (HgCl2) traces by Shaker et al., (2010).

3.1.3. Preparation of Pots

The clay pots of 26cm diameter and 28cm in height were used for raising tomato seedlings.

Before using the clay pots they were washed and sun dried properly. Each pot contained six

6kg soil. Then these pots were aligned according to treatment plan. Each treatment consisted

28
on eight replicates and 40 clay pots were organized in a complete randomized design (CRD).

Out of eight replicates for every treatment only 3 replicates were considered for studies.

Fig: 3.1 Clay pots filled with experimental soil placed in lawn of Ghazi University

3.1.4. Soil Preparation

For experiment the clay pots were filled up with Garden soil for raising tomato plants. The

cow dung was also added/mixed to this soil in the ratio of 3:1 (garden soil and cow dung).

The soil was dried, sieved and removed impurities and stones. Then each pot was loaded up

with equal amount of this readied soil (garden soil + cow dung).

3.2. Seed Sowing and Mercury (Hg) Treatment

After soil sieving and drying process, different concentration of mercuric chloride (HgCl 2)

was added per Kg of pot soil followed by proper mixing. The healthy seeds of uniform size

were sown/planted in each pot. Ten (10) seeds were sown in the depth of 1 inch in each pot

then watered the soil. Each pot was watered/irrigated with tap water depending on need (soil

condition).

3.2.1. Treatment Plan

In experiment five treatments with control were used and organized in Complete

Randomized Design (CRD) and for all treatment including control + mercuric chloride

(HgCl2) treatments 8 replicates comprising of 40 pots were aligned.

29
 • T1=0 (Control) without Hg
• T2=5 mg of Hg/kg of pot soil
• T3=10mg of Hg/kg of pot soil
• T4=20mg of Hg/kg of pot soil
• T5=30mg of Hg/kg of pot soil

3.3. MORPHOLOGICAL AND PLANT BIOMASS PARAMETERS

3.3.1. Total height of Plant

Overall plant height of chosen plants was estimated after harvesting the plants. The

height of plant was measured from stem tip to root endings by using a measuring tape and the

average quantities were noted at last.

Sum of measurements/treatment
__________________________________________________
Mean of plant height (cm) =
Total No. of plants measured/treatment
3.3.2. Shoot Length (cm/plant)

The length of shoot was recorded from stem tip to stem base of every plant per treatment by

using a tape. The smallest shoot length and largest shoot length was taken. Their average

values were recorded.

Sum of shoot measurements/treatment

____________________________________________
Mean shoot length of plan (cm) =
Total No. of plants measured/treatment

3.3.3. Plant Root Length (cm/plant)

The root length of selected plants per treatments was measured carefully using a measuring

tape. The influence of various mercury treatments of on each plant was noted by the

difference between smallest and largest root length and the average values were recorded.

Sum of root measurements/treatment

___________________________________________________
Mean root length of plant (cm) =
Total No. of plants measured/treatment

30
3.3.4. Branches Number /Plant

The plant total number of branches per treatment of tomato was counted at different

concentration of mercury treatment by using formula provided by Uddin et al., (2005).

Sum of number of branches/plant/treatment

__________________________________________________
Average of branches numbers =
Total No. of selected plants/treatment

3.3.5. Leaves Number /Plant

The total leaves number from each mercury treated and control plants was counted.

Their mean values were recorded with the help of formula used by Parvin et al., (2015).

Sum of number of leaves/plant/treatment

_______________________________________________
Average of number of leaves =
Total Number of plants selected

3.3.6. Flowers Number/Plant

The number of flowers on branches of selected plants per treatments was counted. The

average was recorded with the help of formula used by Atta et al., (2013).

Sum of number of flowers/plant/treatment

__________________________________________________
Average of number of flower =
Total Number of plants selected

3.3.8. Leaf Area/Plant (Cm2/Plant)

Leaf area per plant for each treatment was noted with measuring tape. The measurements

were done at the widest and largest part of leaflet. While the total leaf area was assessed by

portable leaf area meter. Following is the formula for leaf area.

Leaf area (cm2) = Length X Width

For measurement the smallest and largest leaf was selected.

31
3.4. ANALYSIS OF PLANT BIOMASS

3.4.1. Fresh Weight (grams/plant)

The plant fresh weight of all organs was measured immediately after harvest. The roots of

freshly harvested plants were washed to remove soil and air dried for some time. For fresh

weight of plant its organs (leaf, stem, and root) were placed on sensitive digital balance to

measure fresh weight. Their average values per plants per treatments were obtained. The

fresh weight of all organs of plant was considered as fresh weight of total/whole plant.

Following formula was used to calculate average values of fresh weights (Pervin et al.,

2015).

Sum of F.W (specific plant organ)/treatment

Average of F.W of specific plant organ = ______________________________________
Total No. of plants weighed/treatment

Sum of F.W (all organs of plant)/treatment

Average of total plant F.W = ____________________________________
Total No. of plants weighed/treatment
3.4.2. Plant Dry Weight (grams/plant)

The method of measuring dry weight per plant is same to the fresh weight except the plant

sample were placed in electric oven. After harvesting, all the plant‘s organs like roots, leaves,

and stems were collected and cut into very tinny and small pieces then placed in envelops.

The envelops were placed in oven and dried for 72 hours at ―70°C‖. After this, these samples

were shifted into desiccators to allow them to normal at room temperature and then dry

weight for each sample were measured by sensitive digital balance. The average values of

plant organs and total plant was calculated by using formula as follows.

Sum of D.W (specific plant organ)/treatment

Average of D.W of specific plant organ = ______________________________________
Total No. of plants weighed/treatment

Sum of D.W (all plant organs)/treatment

Mean total plant D.W = ______________________________________
Total No. of plants weighed/treatment

32
3.5. Appearance Leaf Color (green, yellow or brown leaves)

Different concentrations of mercury (Hg) treatment per plants have impact on leaves-

appearance of plant. Leaf colors modification/alteration is used for chlorotic effects. The

leaves of mercury (Hg) treated plant was compared with control plant leaves (without

mercury treated plant). The alteration in leaf color appearance in response to mercury (Hg)

concentration was studied.

3.5.1. Determination of Chlorophyll Contents (SPAD value)

Leaf chlorophyll level was measured with the help of portable SPAD chlorophyll meter. For

measuring chlorophyll content with SPAD meter, in each plant the chlorophyll was measured

five (5) intervals from five (5) foliage/leaves present at different positions/places on plant

data was taken in average values and studied chlorophyll content.

3.5.2. Heavy Metal Determination

Assessment of mercury up taken treated plants by was completed by using the procedure of

Cargnelutti et al., (2006). Shoot and Root samples were gathered independently, afterward

dried in oven at 650C for about 72 hours because that temperature is adequate to without

expel dampness without warm disintegration. The dried plant samples 0.2 g were powdered

then processed with H2SO4: HClO4 (6:1 v/v) blends at temperature 85°C till white fumes

show up.

The solution was sieved. It was diluted to 50 ml. Atomic Absorption Spectrophotometer was

used for mercury quantification.

3.6. Leaf Proline Contents (As Stress Indicator)

The extraction of Proline and determination of its amount was done by following the method

of Bates et al., (1973). Plant Leaf tissue was uniformed with sulfosalicylic acid. Then

homogenize material (homogenized leaf) was centrifuged for 20 min at 3000rpm × g. After

that the formed supernatant was mixed with ―acetic acid and acid-ninhydrin‖, and then allows

33
it to boil for 1 hour, and then absorbance was examined at 520 nm. Pure proline was used to

make standard curves to determine actual proline in the samples. Spectrophotometer was

used to measure proline.

3.7. Leaf Protein

Leaf protein was evaluated by Bradford method in which a coomassie brilliant blue reagent.

The change in reagent color from red to blue indicated the presence of protein and amount of

protein in plant leaf samples. The quantification of protein is observed at 595nm using

spectrophotometer. The results were compared with standard protein Bovine serum albumen.

3.8. Determination of pH in Tomato Juice

pH of tomato juice per plant per treatment was measured by pH meter (Mukta et al., 2015).

The juice of tomato was taken in test tube and pH per plant per treatment was measured for

mercury treated plants and compared with the control.

3.9. Yield Parameter of Plants

To examine the tomato yield parameters under mercury (Hg) stress following yield

components were assessed.

3.9.1. Numbers of Fruits/Plant

The total number of fruits for mercury treated plants and control plants were counted and

mean values were recorded with the help of formula given as follows.

Sum of number of fruits/plant/treatment

_____________________________________________
Mean=
Total No. of plants selected

3.9.2. Weight of Fruit per Plant

After harvesting the weight of fruit (grams) for selected plant per treatment was measured by

using digital balance and their mean values was taken by using the following formula (Akinci

et al., 2010)

34
 Sum of Total weight of fruits/plant/treatment
______________________________________________________
Mean=
Total No. of plants selected

3.9.3. No of Seeds in Fruit Plant-1 Treatment-1

Seeds were removed from tomato with the help of needle and all seeds were put in sieve,

washed with tap water to remove the flesh then air dried. After that the total number of seeds

of tomato plant per treatment was counted by selecting maximum and minimum fruit size

counted their seeds and average values were taken and compared with control

3.9.4. Fruit Measurement per Plant (width + Length)

Size of fruit of mercury treated plants was measured with measuring tape. The fruits of

maximum and minimum size per mercury treated plant were selected and their average

values were observed.

35
 CHAPTER 4
RESULTS AND DISCUSSION

4.1. Determination of Soil Texture

For the present research work, the soil to be used is analyzed by using the equipment X-Ray

spectrophotometer (PW 1660-Philips). The results from Table: 4.1 demonstrated that the

humified soil exhibit different properties of soil. By investigation of samples of soil displayed

the soil is sandy loam soil containing following percentages such as sand 68.08% and clay

20.66% with pH 7.38. The soils C, N, P, and K contents were measured. Soil carbon (C) was

2.40%, soil nitrogen (N) was 1.38%, soil phosphorus (P) was 17.56% and soil potassium (K)

was 212.56%. The soil electrical conductivity (EC) and organic matter (OM) as well was

found to be 4.08% and 1.71% respectively.

SAMPLE TEXTUE SILIA CLAY EC OM pH C N P K

CLASS SAND

1 Sandy 63.5 20.3 1.64 4.54 7.5 2.7 1.5 17.6 211.0
Loam

2 SL 69.5 20.6 1.71 5.16 7.3 2.8 1.4 17.0 217.1

3 SL 70.6 21.4 1.75 3.64 7.4 2.3 1.3 17.5 207.6

4 SL 67.2 20.6 1.86 3.5 7.4 2.2 1.4 17.4 212.6

5 SL 69.6 20.4 1.63 3.6 7.3 2.04 1.3 18.3 214.5

Table: 4.1 Study of various characteristics of Soil samples

S: Sample EC: Electrical conductivity OM: Organic Matter

K: Potassium SL: Sandy Loam P: Phosphorus
N: Nitrogen C: Carbon

36
4.2. MORPHOLOGICAL AND PLANT BIOMASS ATTRIBUTES

4.2.1. Total Height of Plants

The table: 4.2, shows that mercury (Hg) had significant effect on total height of tomato plants

as compared to control. The decline in total height of plant as compared to control at different

treatments of mercury (Hg) such as T1 11%, T2 27%, T3 39% and T4 56% respectively.

However, the higher level of mercury (Hg) affects the total height of plant more than lower

doses/treatments of mercury.

T0=control T1=5mgHg/kg T2=10mgHg/kg T3=20mgHg/k T4=20mgHg/kg

g) g) )
Fig: 4.1 Tomato plants showing growth under various levels of Hg treatment and
control

4.2.2. Shoot Length

The heavy metal mercury significantly affected the shoot length when compared with control.

High concentrations of mercury metal were proven to influence the length of plant shoot.

Mercury at all concentrations causes significant reduction in shoot length of tomato plants.

The decreased in shoot length was at Hg level T1 8%, T2 22%, T3 34.8%, T4, 51.9%

respectively. The decline in shoot length was 8-51.9% at all treatments T1-T4. Mercury metal

has great influenced on shoot length at highest level (T4) that cause 51.9% reduction.

37
Fig: 4.2. Effect of various treatments of Hg on shoot length

4.2.3. Root Length

Mercury (Hg) has phytotoxic effect (toxic for plants). The length of root was also influenced

by phytotoxic effect of mercury. The length of roots was reduced by all concentrations of Hg

in contrasted with control. The decline in root length of plant in contrast with control at

different concentrations of mercury (Hg) such as T1- 18%, T2- 33%, T3- 51%, T4- 66%. The

reduction in root length of tomato plant at all concentrations T1-T4 (18-66%) as compared to

control. The significant effects of mercury were shown at highest doses (T3 and T4) that

caused 51% and 66% reduction.

38
Table: 4.2 One Way Anova for Growth Parameters.

SOV DS MS F ratio
TP L
Between groups 4 740.6 710***

Within groups 10 1.04

Total 14

Rt L
Between group
4 96.57 269***
Within group
10 0.35
Total
14

St L
Between groups 4 304.4 384***

Within groups 10 0.79

Total 14

ANOVA: Analysis of variance

SOV: Source of variants is Parameters
TP L: Total Length of Plant
Rt L: Root length
St L: Shoot length
DF: Degree of freedom
MS: Mean square
***: p-value is highly significant

39
TABLE: 4.2.1 Mean values for growth parameters of tomato plants at Hg treatments.

Hg mg/kg TP L (cm) Rt L (cm) St L (cm)

0 (Control) 70.1 21.6 48.5

5 62.2 17.7 44.5

10 51.0 14.3 36.6

20 42.1 10.5 31.6

30 30.56 7.267 23.3

70

60
% REDUCTION VS CONTROL

R² = 0.9536

50

40
Rt L
30
St L
20 TP L
10

0
T0 T1 T2 T3 T4
-10
Hgmg/kg

Fig: A shows the effect of Hg on total height of plant, shoot length and root length of tomato
plants

40
4.2.4. Numbers of Branches /Plant/Treatment

The branches of tomato plants are not affected by normal soil medium (soil without mercury,

control). While the numbers of branches are influenced by mercury treatments. When the

concentration of mercury (Hg) in soil increased there occur gradual decreased in number of

branches at all treatments of mercury. Table: 4.3 / Figure B of study data shows that Hg has

significant impact on the numbers branches of tomato plants at high concentrations in

contrast to control. Declined in branches number/plant per treatment was at Hg level T1

4.76%, T2 14%, T3 23.8%, T4 33% respectively. However higher doses of mercury has great

impact on number of branches than lower doses of Hg.

Fig: 4.3 Effect of Hg treatments on the numbers of branches

41
4.2.5. Numbers of Leaves/Plant/Treatment

The leaves are the photosynthetic organ of plants. The numbers of leaves per plant per

treatment decreased as compared to control due to Hg contaminated soil. The Hg decreases

the numbers of leaves at all Hg treatments (5-30mg/kg). So the mercury influenced the

numbers of leaves of tomato plants. Table: 4.3/ Figure C illustrates that the mercury has

significant impact on the numbers of leaves of tomato plants. The numbers of leaves reduced

at low and high doses of Hg. The reduction in numbers of leaves in contrast to control was at

mercury concentrations T1 4.8%, T2 13.8%, T3 23%, T4 34% respectively.

Fig: 4.4 Numbers of leaves under Hg treatment.

42
 Table: 4.3 One Way Anova for Numbers of Branches/Plant and Leaves/Plant.

SOV DF MS F ratio

N.O.Br/P

Between groups 4 2.733

6.83**
Within groups 10 0.40

Total 14

N.O.L/P

Between groups 4 96.90

72.7***
Within groups 10 1.33

Total 14

ANOVA: Analysis of variance N.O.Br/P: Numbers of branches per plant

SOV: Source of variants (Parameters) N.O.L/P: Numbers of leaves per plant

DF: Degree of freedom **: Less significant

MS: Mean square

***: Highly significant

Table: 4.3.1 Mean Values for Numbers of Branches/Plant and Leaves/Plant

Hgmg/kg N.O.Br/P N.O.L/P

0 (control) 7.0 41.0

5 6.6 39.0

10 6.0 35.3

20 5.3 31.3

30 4.6 27.0

43
 35
% REDUCTION VS CONTROL

30
25 R² = 0.8894

20
15
10
Reduction in
5
Numbers of
0 branches
T0 T1 T2 T3 T4
-5
-10 Hgmg/kg

Fig: B shows the effect of Hg on numbers on branches in tomato plants.

40
35 R² = 0.8779
30
25
20
Reduction
15 in leaves
10 no's

5
0
-5 T0 T1 T2 T3 T4

-10
Hgmg/kg

Fig: C shows the effect of Hg on numbers of leaves in tomato plants.

44
4.2.6. Numbers of Flowers Plant-1 Treatment-1

The flowers are reproductive part of plant. The tomato plant is bisexual plant. The flowers of

tomato are called perfect flowers because both organs are found on same flower. When

contrasted with control the cultivar Roma showed reduced number of flowers grown under

mercury contaminated soil. Hg at lower doses the phytotoxic effect on flowers was less than

higher doses. As the concentration of Hg increases, its phytotoxic effect becomes more. So

the more decline in flowers number each plant. The decreased in numbers of flowers of Hg

treated plant at all level T1-T4 was 18%-63.6%. Tomato flowers are sensitive to mercury

pollution. Hg has influenced on the numbers of flowers as compared to control, the decline in

numbers of flowers was at Hg level were T1 18%, T2 27%, T3 40.9% and T4 63.6% as

demonstrated in figure D..

Table: 4.4 One Way Anova for Numbers of Flowers.

SOV DF MS F ratio
N.O.F/P 10.7 **
Between groups 4 9.266
10 0.86
Within groups
14
Total

ANOVA: Analysis of variance

SOV: Source of variants (Parameters)

DF: Degree of freedom

MS: Mean square

**: Less significant

N.O.F/P: Numbers of flowers per plant

45
Table: 4.4.1 Mean Values for Numbers of Flowers

Hgmg/kg N.O.F/P
T0 control 7.33
T1 5 6.0
T2 10 5.33
T3 20 4.33
T4 30 2.66

70

60
% REDUCTION VS CONTROL

50
R² = 0.9067
40

30

20 Reduction in No's of
flowers
10

0
T0 T1 T2 T3 T4
-10
Hg mg/kg

Fig: D shows the decline in numbers of flower with increasing concentration of Hg.

46
4.2.7 Growth Tolerance Index (G.T.I %)

Tolerance index is a parameter that is used to identify the tolerance or sensitivity of plant.

The growth tolerance index, in relation to plant height at control (soil without Hg treatment)

was 100% .Table: 4.5 and Figure F shows decrease in tolerance index (plant height) at all

mercury treatments against 5- 30 mgHg/kg. The decrease in tolerance index was 11%-56%.

Hg has significant influenced on the total length of plants (growth). (Akinci et al., 2010)

4.2.8. Plant Leaf Area by Portable Leaf-Area-Meter

The leaf area was recorded by portable leaf area meter during flowering stage of tomato

plants. There was significant reduction in leaf area of all Hg treated plants when compared

with control. The decline in leaf area was at T1 2.4% reduction and at T2 5.5%.while highest

reduction in leaf area was at high Hg concentrations (T3 20mgHg/kg-T4 30mgHg/kg) 20-

29.9% as presented in Table: 4.5 and Figure E.

47
Table: 4.5 One Way Anova for Leaf Area and Growth Tolerance Index

SOV DF MS F ratio
L.A
Between groups
4 1453.79 2484***
Within groups
10 0.59
Total
14

G.T.I
Between groups 4 1489.86 5912***
Within groups 10 0.25
Total 14

ANOVA: Analysis of variance DF: Degree of freedom

MS: Mean square L.A: Leaf area

G.T.I: Growth Tolerance index ***: p-value is highly significant

SOV: Source of variants (Parameters)

Table: 4.5.1 Shows for Mean Values of Leaf Area and Growth Tolerance Index

Hgmg/kg L.A (cm2) G.T.I (%)

0 158.56 100.00
5 162.42 88.73
10 149.71 72.70
20 126.87 60.00
30 111.03 44.00

48
 35

% REDUCTION VS CONTROL 30 R² = 0.7456

25
20
15
10 Leaf area reduction
5
0
T0 T1 T2 T3 T4
-5
-10 Hgmg/kg

Fig: E Assessment of leaf area (cm2) at all Hg levels

60
R² = 0.9267
REDUCTION VS CONTROL %

50

40

30 T.I

20

10

0
T0 T1 T2 T3 T4
-10
Hgmg/kg

Fig: F Presented the Tolerance index of tomato plant at Hg treatments.

49
4.3. ANALYSIS OF PLANT BIOMASS

4.3.1. Determination of Fresh Weight (g plant-1)

The influence of mercury on fresh weight of roots and shoots per plant per treatment was

measured in gram. In comparison with Hg treated plants, the fresh weight of control tomato

plants (grown in soil without Hg contamination) remain unaffected. There was a noteworthy

fresh weight reduction in at all mercury treatments.

4.3.2. Fresh weight of shoot plant-1 treatment-1

The fresh weight of shoot reduced against Hg concentrations (5, 10, 20 and 30mgHg/kg)

when contrasted with control. The shoot fresh weight of was declined to 4.9% at T1, 11.4% at

T2, 21.3% at T3 and 33.9% at T4 respectively in comparison with control. The reduction in

shoot fresh weight was the phytotoxic effect of heavy metal Hg. The Hg imposes significant

effect in shoot fresh weight at higher dose than lower dose of Hg.

4.3.3. Fresh weight of root plant-1 treatment-1

Table: 4.6 and figure G of the study data shows that Hg has affected significantly the fresh

weight of root in tomato plants. The phytotoxic effect of Hg at the doses T1-T4 (5-

30mgHg/kg) causes reduction in root fresh weight. The drop in root fresh weight had greatly

influenced by high doses of mercury (Hg) than low dose of Hg. The reduction in root fresh

weight was at T1 8%, T2 19%, T3 28%, T4 45% respectively.

4.3.4. Fresh Weight of Total Plant

The decline in fresh weight of total plant was measured in figure G and Table: 4.6 shows that

Hg has significant phytotoxic impact on fresh weight of total plant. The plant weight reduced

was 5.8%, 13.6%, 23.3% and 37% at T1- T4 respectively when compared with control.

50
4.4. Determination of dry weight (g/plant)

4.4.1. Dry weight of shoot plant-1 treatment-1

The shoot dry weight of tomato plants reduced at all Hg treatments (5-30mgHg/kg). The

shoot reduced their weight by 12%, 19%, 46% and 67.7% at concentrations (T1, T2, T3 and

T4) respectively. As compared to control there was significant reduction in dry weight of

shoot of Hg treated plants. The decline in dry weight increased with respect to increasing

levels of Hg.

4.4.2. iDry iweight of root iplant-1 itreatment-1

The root reduced its dry weight against different Hg concentrations (5-30mgHg/kg) by

37.3%, 63.97% 70.8%, and 82.9% at Hg concentrations T1, T2, T3 and T4 respectively than

control. The root dry weight reduction represents the phytotoxicity of Hg that has

significantly affected on root dry weight.

4.4.3. Total plant dry weight

Table: 4.6 and figure H of study data shows that itotal iplant idry iweight of tomato was

reduced to highly significant level. The itotal iplant idry iweight decline was 20.4% at T1,

33.6% at T2, while the maximum reduction was showed at high doses of mercury

20mgHg/kg and 30mgHg/kg (T3 & T4) was 54.21% and 72.59% respectively.

51
Table: 4.6 One Way Anova for Various Characteristics of Plant Biomass.

SOV DF MS F ratio
St. F.W
Between groups 4 114.14 289***
10
Within groups 0.39
14
Total
Rt. F.W
Between groups 4 32.74 33.7***
10
Within groups 0.97
14
Total
TP. F.W
Between groups 4 268.55 624***
10
Within groups 0.43
14
Total

St. D.W
Between groups 4 54.41 400***

Within groups 10 0.13

Total 14

Rt. D.W
Between groups 4 17.45 217***

Within groups 10 0.08

Total 14
TP. D.W
Between groups 4 125.52
3633***
Within groups 10 0.03

Total 14

52
ANOVA: Analysis of variance

SOV: Source of variants (Parameters)

DF: Degree of freedom

MS: Mean square

***: Highly significant

St. F.W: Shoot fresh iweight

Rt. F.W: Root ifresh weight

TP. F.W: Total plant fresh weight

St. D.W: Shoot idry weight

Rt. D.W: Root dry iweight

TP. D.W: Total plant dry weight

Table: 4.6.1 Mean Values for Plant Biomass

Hg mg/kg St. FW Rt. FW TP. FW St. DW Rt. DW TP.DW

Control 45.5 18.76 64.26 15.5 7.31 22.8

5 43.26 17.23 60.50 13.6 4.55 18.1

10 40.30 15.2 55.46 12.5 2.63 15.1

20 35.80 13.46 49.26 8.3 2.13 10.4

30 30.06 10.30 40.36 5.0 1.25 6.25

53
 50
% REDUCTION VS CONTROL

40 R² = 0.8811

30
St FW
20 Rt FW
TP FW
10

0
T0 T1 T2 T3 T4
-10 Hgmg/kg

Fig: G Assessment of shoot, root, and total plant fresh weight of tomato under Hg toxicity.

90
% REDUCTION VS CONTROL

80
R² = 0.99
70

60

50 Rt DW
40
St DW
30
TP DW
20

10

0
T0 T1 T2 T3 T4
Hgmg/kg

Fig: H Assessment of root, shoots, and total plant Dry weight of tomato under Hg toxicity.

54
4.5. BIOCHEMICAL ANALYSIS

4.5.1. iDetermination of Chlorophyll Contents (iSPAD Value)

The content of chlorophyll was measured by SPAD chlorophyll meter 502 japan. The

chlorophyll content is influenced by Hg treatments. When compared with control plant the

i chlorophyll icontent was less in Hg treated plants. When the concentration of mercury metal

increased, the decline in chlorophyll content also increased. According to table: 4.7 and

Figure I, there were significant reduction percentages from T1-T4 (15.4%-43.8%) as

compared to control. The decline at level T1 was 15.4%, T2 27.3%, T3 35.9% and T4 43.8%

respectively.

4.5.2. Uptake of Mercury Heavy Metal Determination

In Root

A remarkable uptake by root of tomato showed at all Hg level. There was no mercury

traced observed in tomato plants grown-up in soil without Hg contamination. The uptake

of mercury by roots was 1mg at T1, 2.7mgHg/kg at T2, 4.1mg at T3 and 5.5 mg at T4

respectively. The accumulation of Hg was increased with increasing concentrations.

In Shoot

The mercury uptake by shoot was lower than root. At 5mgHg/kg (T1) there was no uptake by

shoot. At 10mgHg/kg and 20mgHg/kg (T2 and T3) mercury uptake was 0.5 mg and at

30mgHg/kg T4 0.8 mg uptake.

In Leaf

The uptake of mercury by tomato leaf was not detectable at all treatments. Table: 4.7 and

Figure J revealed that significant accumulation of mercury in roots.

55
Table: 4.7. One Way Anova for Plant Biochemical Analysis

SOV DF MS F ratio
SPAD
Between groups 4 205.82 65.8***
Within groups 10 3.129
Total 14

St Hg
Between groups 4 0.369 61.5***
Within groups 10 0.006
Total 14

Rt Hg
Between groups 4 15.20 2534***
Within groups 10 0.006
Total 14

Lf Hg
Between groups 4 0.00 0.00
Within groups 10 0.00
Total 14

56
Table: 4.7.1 Mean values of chlorophyll and mercury metal accumulation.

Hgmg/kg SPAD value Hg uptake in Shoot Root

leaf
0 47.9 0.0 0.0 0.0

5 40.5 N.D N.D 1mg

10 34.8 N.D 0.5 mg 2.7 mg

20 30.6 N.D 0.5 mg 4.1 mg

30 26.9 N.D 0.8 mg 5.5 mg

50
45
% REDUCTION VS CONTROL

R² = 0.992
40
35
30
25
20 SPAD value
15 decreased
10
5
0
-5 T0 T1 T2 T3 T4

Hgmg/kg

Fig: I Analysis of SPAD value under phytotoxic Hg

57
 6

5 R² = 0.8098
% REDUCTION VS CONTROL

4
Lf
3
St
2
Rt
1

0
T0 T0 T0 T1 T1 T1 T2 T2 T2 T3 T3 T3 T4 T4 T4
-1

-2

-3 Hgmg/kg

Fig: J Determination of Hg metal accumulation in root, stem and leaves

58
4.6. Leaf proline contents (as stress indicator)

The toxic effect of mercury metal (pollutant) was assessed by proline content of the leaf.

Proline content is sensitive to Heavy metal stress therefore this parameter is used as stress

indicator. As compared to control (soil without Hg pollution) the leaf proline content in

tomato plants increased with increasing concentrations of Hg. The leaf proline content

increased at all Hg levels T1-T4. Increase in Proline content when compared with control was

at T1 1.0%, T2 6.7%, T3 35.7% and T4 47.35% respectively as shown in Table: 4.8 and

Figure k.

4.7. Leaf Protein (ug/g)

According to the study data of Table 8 and Figure k, the Total soluble protein in leaves of

tomato plant decreased at all Hg concentrations as compared to control. High doses of Hg

influenced more greatly on total soluble leaf protein than lower doses. The leaf protein

reduction was 4.3%, 15.98%, 21.96% and 56% at T1, T2, T3 and T4 respectively. Total

soluble leaf protein was decreased significantly in tomato plants.

4.8. Tomato Fruit Juice pH

The pH of tomato juice was also affected by mercury toxicity. At high concentration of

mercury the pH of tomato juice decreased due to Hg phytotoxicity. As compared to control

the pH becomes more acidic at high Hg doses 20-30mgHg/kg. The tomato fruit reduced its

pH at all treatments T1, T2, T3, T4 was 14.9%, 22.38%, 38%, and 47.76% respectively.

59
Table: 4.8 One Way Anova for Analysis of Proline, Protein, Leaf Color and Tomato pH

SOV DF MS F ratio
Leaf proline
Between groups 4 150.491 649***
Within groups 10 0.232
Total 14

Leaf protein
Between groups 4 64186.9 53161***
Within groups 10 1.2
Total 14

No. of leaf color

appearance 4 29.10 48.5***
Between groups 10 0.60
Within groups 14
Total
Tomato pH
Between groups 4 2.126 103***
Within groups 10 0.02
Total 14

Table: 4.8.1 Mean Values of Leaf Proline, Protein, Leaf Color Appearance, Tomato pH

Hgmg/kg Leaf proline Protein ug/g Leaf color pH of tomato

umole/g appearance juice

0 18.26 734.80 0 4.46

5 18.06 702.47 0 3.80

10 19.5 617.37 2 3.46

20 24.8 573.43 5 2.76

30 34.7 321.17 7 2.33

60
 60
REDUCTION VS CONTROL %

50 R² = 0.9486

40
Proline
30
pH
20 protein

10 Log. (pH)

0
T0 T1 T2 T3 T4
-10
Hgmg/kg
Fig: K Demonstrates effect of Hg on leaf proline, protein, and tomato juice pH

61
4.9 Yield parameter of plants

4.9.1 Numbers of fruits per plant

According to data of study of Table 9 and Figure showed a significant reduction in fruits of

tomato per plant at all mercury treatments. The decreased in numbers of fruit at Hg

concentrations 5-30mgHg/kg (T1-T4) as compared to control (Hg free soil) was 14%, 28.5%,

52.3% and 66.6% respectively. This decline in fruit numbers was due to Hg toxicity.

Fig: 4.5 Numbers of fruit per treatment.

4.9.2. Weight of Fruits per Plant

The fruit weight of tomato plant was reduced as compared to control due Hg phytotoxic

effect. The reduction in fruit weight against Hg treatments was 7.9% at T1, 19% at T2, 30.7%

at T3 and 46.7% at T4. Highest concentration of mercury causes highest reduction in fruit

weight (46.7%). The Hg causes significant reduction in fruit weight of tomato plant.

62
Fig: 4.6 fruit weight by digital weighing balance.

4.9.3 No. of seeds in fruit plant-1 treatment-1

As compared to control, for ieach iselected fruit per plant total numbers of seeds were counted

and they were found reduced seed numbers. The continuous decreased in numbers of seed of

fruit per plant at all mercury concentrations T1-T4. At 5-10mg/kg of Hg reduction in seeds

per fruit per plant was 13% and 24% and at 20-30mg/kg of Hg numbers of seeds reduced to

33.2% and 39% (Table 9/ figure L)

Fig: 4.7 seeds numbers per treatment

63
4.9.4 Fruit Measurement per Plant (width+Length)

As compared to control the fruit size was measured and found affected by Hg treatments at

all doses. Fruit girth/size was decreased continuously at all treatments of Hg. The decline in

fruit girth/size at T1, T2, T3 and T4 was 14%, 23.8%, 32.79% and 47% respectively.

Fig: 4.8 Effect of Hg on fruit size.

64
Table: 4.9 One Way Anova for Yield Parameters.

SOV DF MS F ratio
N. O. F/P
Between groups 4 10.90 14.9***
Within groups 10 0.73
Total 14

W. O. F/P
Between groups 4 238.35 59.8***
Within groups 10 3.98
Total 14

N. O. S/F
Between groups 4 549.95 1677***
Within groups 10 0.32
Total 14

F. S/P
Between groups 4 26.01 51.7***
Within groups 10 0.50
Total 14

ANOVA: i Analysis of variance

SOV: Source of ivariants (Parameters)
DF: Degree of ifreedom
MS: Mean isquare
***: p-value is highly significant
N. O. F/P: Numbers of fruits per plant
W. O. F/P: Weight of fruit per plant
N. O. S/F: Numbers of seeds per fruit
F. S/P: Fruit size per plant

65
Table: 4.9.1 Mean values of yield parameters of tomato plants.

Hgmg/kg N. O. F/P W. O. F/P (g) N. O. S/F F. S/P(W+L)

0 7 48.16 86.43 16.41

5 6 44.33 74.83 14.08

10 5 38.66 65.50 12.50

20 3 33.33 57.70 11.03

30 2 25.66 52.63 8.66

80
% REDUCTION VS CONTROL

70 R² = 0.9127
60
50
40 N.O.F
30 W.O.F
20 F.S
10 N.O.S
0
-10 T0 T1 T2 T3 T4

-20 Hgmg/kg

Fig: L Presented yield parameters (numbers of fruits, weight of fruits, fruit size, numbers of
seed/fruit) affected by Hg toxicity.

66
 DISCUSSION
According to valiappan et al., 2002 the plant growth is effected by mercury metal. The

mercury is toxic metal that has negative impact on the plant physiology if its low dose is

present in soil. For understanding the effect of mercury metal on tomato plants a pot

experiment was performed to check its growth and development under different Hg doses (5,

10, 20 and 30mg//kg). The total plant height was reduced to 11-56% from T1-T4

respectively. The significant decreased in plant growth was R² = 0.9536 mentioned in table 2

and figure A. The decline in root length at lowest dose 5mg/kg was 18%, at 10mg/kg the

reduction was 33%, highest decrease in root length was 51% and 66% at doses 20, and

30mg/kg of Hg. Similar findings was also described by Gupta et al., 2013 that Hg, As, and Pb

accumulate in roots and causes reduction in root length. Mercury has negative effect of on

shoot length and decreased the shoot growth at all doses was 8%, 22%, 34.8%, 51.9% from

5-30mg/kg respectively. Mercury has more influenced negatively on root growth than shoot

growth. The length of root was adversely affected than shoot length.

Umadevi et al., 2009 also investigated the effect of phytotoxic mercury on plant growth using

Hg doses 0.05-0.1mM and reported that Hg badly impact on length of root than shoot length.

Significant effect of mercury on roots indicates that Hg localization find in roots as compared

to shoot. Hg is absorbed and accumulated in roots differently therefore the roots show

different responses to Hg. The high level of Hg accumulated in roots is due to roots have the

ability to absorb and detoxify Hg rapidly (Shaukat et al., 1999).

Mishra and Choudhuri (1998) investigated the influence of Hg and Pb on rice varieties and

reported that toxicity of mercury is more than lead. The mercury has significant effects on

growth of plant, mercury decreased the growth of root and shoot. According to Cardenaz-

Hernandez et al., 2009 Heavy metals influenced negatively on plant growth and

development. iRoot igrowth was reduced to 50% due to Hg stress in Zea mays L. (Ivanov et
67
al., 2013). Patra et al., 2004 reported the permeability of cell membrane is affected because

Hg causes restriction of water channels in plants that lead to growth retardation.

Our findings were the numbers of leaves were affected significantly at high concentration

upto 34% reduction in leaf number at 30mg/kg. The increased in browning of leaves with

increasing concentrations of Hg. Similar findings in numbers of leaves reduction and

cholorotic patches were reported under mercury phytotoxicity by Malar et al., 2015.

A physiological factor that is used to indicate plant responses to different biotic and abiotic

stress is leaf area. The area of leaf decreased in response to Hg stress. The maximum

reduction in leaf area was observed at high concentration of Hg 20 and 30mg/kg about 20 to

30% respectively as compared to control (Table: 4.5 and figure E). Comparable results were

observed from previous literature that satisfies the values regarding decline in leaf area

(Marrugo-Negrete et al., 2016). The reduction in surface of leaf was totally depend on

mercury metal concentration. High level of Hg reduced the area of leaf demonstrated by

Mondal, 2015; Faizan et al., 2011; Anayat et al., 2014.

Photosynthesis performs important for the biomass yield of plant. The biomass attributes

i (root and shoot fresh/dry weight) were examined in tomato plants under Hg toxicity. Both the

plant fresh weight and dry weight are significantly affected by high doses of mercury 20-

30mg/kg. The fresh weight of plant was decreased 5.8% - 37% from 5-30mg/kg of mercury.

The decline in fresh weight increased with increasing the Hg doses at 20mg/kg of Hg the

decline was 23.3% and at 30mg/kg of Hg the weight reduced to 37%. Table: 4.6 and Fig G

presented the significant (R² = 0.8811) effect of Hg on plant fresh weight. Plant Dry weight is

also influenced by Hg stress. The Dry weight was reduced 20%, 33.6%, 54% and 72.5% at

Hg doses 5-30mg/kg. Table: 4.6 showed the toxic effect of Hg on plant dry weight. The

Mercury (HgCl2 ) in plant ‗soya bean‘ decreased the fresh weight as well as dry weight

68
(Palanisamy, 2012). Mercury in peanut seedling reduced the fresh weight is based on

concentration (Bhanumathi, 2005). Mercury metal causes reduction in dry weight of

seedlings cucurbita pipo due to its toxicity (Bankji et al., 2018). Wheat plants at 25uM of Hg

reduced its weight by 30% of total plant fresh weight (Sahu, 2012). Upto 50% of biomass

lost when Jatropha curcas was exposed to Hg (Marrugo-Negrete et al., 2016). Similar results

were evaluated by Shiyab et al., 2009; Morales and Gallego 2013 for different plants

(Brassica juncea and Brachiaria dictyoneura respectively).

Table 5 and figure F represented significant reduction in tolerance index was R² = 0.9267 as

compared to control was 11%, 27.3%, 40% and 56% at 5, 10, 20 and 30mg/kg respectively.

The literature reported the same finding. Seedlings of Albizia lebbeck (L.) were treated with

HgCl2 at concentrations 1, 3, 5, 7 Mm possessed declines in tolerance percentage 48.35% at

high Hg dose (7mM) as compared to control where tolerance percentage was 100%. (Iqbal,

2014). Similarly the effect of Hg on the Vigna radiate (L.) plant seedling have the same

finding, Hg also caused highly significant reduction in tolerance index 14%-65.8% at all the

doses 1, 3, 5, 7 mM (Iqbal et al., 2015). The findings were comparable to our research that

growth tolerance reduced to 42%–46% at 15 mg L-1 of Hg (Hu et al., 2020).

Photosynthesis is important parameter to find the plant stress. It sometimes act as biomarkers

for plants (Pinheiro et al., 2013). Mercury effects the photosynthetic pigments and disturbed

the rate of photosynthesis by replacing metal ions in pigments (kupper et al., 1998). Hg is

phytotoxic metal for plants and had great influenced on plant chlorophyll level. The high

doses of Hg cause increased in decline percentage. The chlorophyll reduction ranged from

15.4%-43.8% from the doses 5-30 mg/kg. Table: 4.7 and Figure I interpreted the toxic effect

of Hg on chlorophyll content of tomato plants. Previous studies also revealed that Hg at high

concentration 75uM applied on fagopyrum tataricum showed 70.3% decline in chlorophyll as

compared to control during 1 month of exposure and lower decline was 68% at 15 days

69
Pirzada et al., 2018. In the same way chlorophyll level in wheat was negatively influenced by

Hg at high doses (Sahu, 2011)

Proline is used as stress indicator. In case of abiotic stress like heavy metals, its level

increased based on concentration dependent. Table 8 and Figure K presented proline content

increased significantly (R² = 0.9486) ranging from 1% at 5mg/kg, 6.7% 10mg/kg, 35.7% at

20mg/kg and 90% at 30mg/kg 47%. Phytotoxicity of Hg and Cd effect the Gram leaf proline

content in at level 10 and 25umole/L of both Hg and Cd metal. There was 38% increase in

proline content at 25umole/L of Hg dose. (Tantrey and Agnihotri, 2010). Proline level

increased with increasing Hg doses in Spirodela polyrhiza (L.) by (Singh, 2020). Previous

literature shows that more proline content of leaf was accumulated due to Hg stress as

compared to pb (Hamim et al., 2019).

Leaf total soluble protein decreased with increasing concentrations of Hg, the reduction in

leaf protein was highly significant (R² = 0.9486) ranged 4%-56% from 5-30mg/kg as

compared to control (Table: 4.8 and Figure K). Similar results were reported by other

researchers, due to phytotoxic effect of mercury, the total soluble leaf protein was decreased

by 51.35% at high dose. (Malik et al., 2018). Gauba et al., 2005 reported decreased in total

soluble protein due to Hg toxicity at highest dose (2mM of HgCl 2) was 64%. Soluble protein

decreased with increasing dose and time duration in cucumber (Cargnelutti et al., 2006).

Decreased in leaf protein was highest (19%) at high dose of mercury (Sahu, 2011).

The Hg has significant influenced on yield in tomato plants. As compared to control, the

numbers of tomato fruits decreased at all levels of mercury. The highest decreased in fruit

numbers about 66.6% was occurred at highest dose (30mg/kg) and at lowest dose (5mg/kg)

there was only 14% decline in fruit numbers table: 4.9 and figure L represented significant R²

= 0.9127 yield retardation in tomato plants. The decreased in yield of white cabbage under

70
Hg toxicity was observed by Rolka et al., 2018. Xu et al., 2020 reported ginger growth was

stopped by exposure of Hg for long duration. The decline in yield was 26% at treatment of

9mg/kg. The significant reduction was 25% in yield under Hg stress at high dose 150mg/kg

by Ceicko et al., 2007. Comparable consequences were also observed by Ansari et al., 2009

mustard yield was declined (25% and 37%) by Hg at treatments (6 and 12 mg/kg). HgCl2

causes 32% reduction in yield in oats plants investigated by Popa et al., 2007

The accumulation of Hg in occurred at all levels in tomato roots (5-30mg/kg). Excessive

amount of Hg metal was accumulated at highest concentrations (at 30mg/kg 5.5mgHg/kg) in

roots than stem. Gao et al., 2010 and Lomonte et al., 2010 also contributed to our findings

that the assessment of mercury was higher in roots in Jatropha curcus and Atriplex

codonocarpa. The highest concentration of Hg and Si was found in ginger root, then rhizome

and least in stem and leaf. (Xu et al., 2020). Hg available in high concentration in soil and

roots and low in shoot reported by Perez-Sanz et al., 2010. Similar findings were observed

that highest level of Hg mercury detected in roots. The mercury accumulation was

concentration dependent reported by Zornoza et al., 2010; Wang and Greger, 2004; Sahi et

al., 2006

71
 CONCLUSION

Current study indicates that the heavy metal mercury is phytotoxic for tomato plants. The

toxicity of Hg influenced significant impacts on different parameters of Roma tomatoes at

treatments (5, 10, 20 and 30mg/kg of Hg). The numbers of leaves, and leaf surface/area also

decreased due to Hg stress. The toxic influence of Hg also changes leaf color appearance.

Roots are most sensitive to mercury stress the iroot length, root fresh weight and dry weight

decreased due to Hg toxicity. The yield is reduced under Hg stress. Low concentrations at 5

and10mg/kg of Hg do not imposes significant effects on plant growth, biomass, yield,

chlorophyll content but highly significant effects influenced at high level of mercury(20 and

30mg/kg). Current study revealed the accumulation of mercury in roots is higher than stem.

72
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